U.S. patent application number 09/225426 was filed with the patent office on 2002-06-20 for purified nitric oxide synthase.
Invention is credited to CHEN, YIJUN, ROSAZZA, JOHN P.N..
Application Number | 20020076782 09/225426 |
Document ID | / |
Family ID | 33455983 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020076782 |
Kind Code |
A1 |
ROSAZZA, JOHN P.N. ; et
al. |
June 20, 2002 |
PURIFIED NITRIC OXIDE SYNTHASE
Abstract
The present invention provides a novel constitutive nitric oxide
synthase (NOS) that utilizes both L-arginine and arginine-rich
peptides, oligopeptides or proteins, e.g., bradykinin (BK), as
substrates in the synthesis of nitric oxide (NO). Also provided are
methods of controlling, regulating or modulating NO synthesis in a
subject.
Inventors: |
ROSAZZA, JOHN P.N.; (LOWA
CITY, IA) ; CHEN, YIJUN; (LOWA CITY, IA) |
Correspondence
Address: |
HEIDI S NEBEL
ZARLEY MCKEE THOMTE VOORHEES & SEASE
801 GRAND AVENUE, SUITE 3200
DES MOINES
IA
503092721
|
Family ID: |
33455983 |
Appl. No.: |
09/225426 |
Filed: |
January 5, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09225426 |
Jan 5, 1999 |
|
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08675821 |
Jul 5, 1996 |
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5856158 |
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Current U.S.
Class: |
435/191 ;
424/94.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 9/0075 20130101; C07K 7/18 20130101; C12Y 114/13039
20130101 |
Class at
Publication: |
435/191 ;
424/94.1 |
International
Class: |
C12N 009/06; A61K
038/43 |
Claims
What is claimed is:
1. A method of regulating or controlling nitric oxide production in
a mammalian subject comprising administering to the mammal a nitric
oxide-regulating amount of an arginine-rich peptide, oligopeptide,
or protein inhibitor of nitric oxide synthase.
2. The method of claim 1, wherein the nitric oxide synthase is
nNOS-II.
3. The method of claim 1, wherein the inhibitor is
N.sup.G-methyl-L-argini- ne.
4. The method of claim 1, wherein the inhibitor is
N.sup.G-nitro-L-arginin- e.
5. The method of claim 1, wherein the inhibitor is a peptide or
oligopeptide.
6. The method of claim 1, wherein the nitric oxide production is
increased.
7. The method of reducing the rate of nitric oxide production in a
mammalian subject comprising administering to the mammal a nitric
oxide inhibiting amount of a peptide, oligopeptide, or protein
inhibitor of the nitric oxide synthase of claim 1.
8. The method of reducing the rate of nitric oxide production in a
mammalian subject comprising administering to the mammal a nitric
oxide inhibiting amount of a peptide, oligopeptide, or protein
inhibitor of the nitric oxide synthase of claim 2.
9. The method of claim 1, wherein the nitric oxide production is
decreased.
10. A method of preventing or treating a nitric oxide-mediated
disease or condition in a mammalian subject comprising
administering to the subject in need of such prevention or
treatment a therapeutically effective amount of a peptide,
oligopeptide or protein inhibitor of nitric oxide synthase.
11. The method of claim 10, wherein the nitric oxide synthase is
nNOS-II.
12. The method of claim 10, wherein the nitric oxide synthase is a
mammalian brain-derived nitric oxide synthase (NOS) protein
purified to an activity at least 6,360-fold, said protein having a
denatured molecular mass as determined by sodium dodecyl sulfate
polyacrylamide gel electrophoresis under reducing conditions of
about 105 kD, and a native homodimeric molecular mass as determined
by gel filtration of about 230 kD), requiring FAD, FMN, Ca.sup.2+
and tetrahydrobiopterin cofactors for the production of nitric
oxide either from L-arginine, or an analog or derivative thereof,
or from an arginine-rich peptide, oligopeptide, or protein
substrate.
13. The method of claim 10, wherein the inhibitor is
N.sup.G-methyl-L-arginine.
14. The method of claim 10, wherein the inhibitor is
N.sup.G-nitro-L-arginine.
15. The method of claim 10, wherein the inhibitor is a peptide or
oligopeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of copending
application Ser. No. 08/675,821, filed Jul. 5, 1996, the disclosure
of which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is directed to a purified enzyme and,
more particularly, to a novel, purified, constitutive mammalian
nitric oxide synthase (NOS) that utilizes both L-arginine and
arginine-rich peptides, oligopeptides (e.g., bradykinin (BK)), and
proteins as substrates.
[0004] 2. Description of Related Art
[0005] Nitric oxide synthases (NOS, EC 1.14.23) are important
enzymes, which convert L-arginine to L-citrulline and nitric oxide
(NO). Nitric oxide is a very short-lived free radical, which is
rapidly oxidized to nitrite (NO.sub.2) and nitrate (NO.sub.3) which
are measured as the stable inactive end products of nitric oxide
formation. The significance, however, lies in the fact that NO
appears to play a pivotal role in a wide variety of physiological
and pathological processes in mammals. These processes include
vasodilation and regulation of normal vascular tone, inhibition of
platelet aggregation, neuronal transmission, cytostasis,
hypotension associated with endotoxic shock, inflammatory
response-induced tissue injury, mutagenesis, and formation of
carcinogenic N-nitrosamines (Nathan, FASEB J. 6:3051-3064 (1992);
Kiechle et al., Am. J. Clin. Pathol. 100:567-575 (1993)). For
example, it is well-known in the art to treat humans afflicted with
angina distress and cardiovascular disease with nitroglycerin,
which acts as a vasodilating agent. In the body, nitroglycerin is
converted to nitric oxide (NO), which is the pharmacologically
active metabolite. See, Palmer et al., Nature 333:664-666 (1988).
Thus, evidence that NO mediates functions as diverse as those which
occur in the brain, the endothelium and the blood, has led to
intense study into the biological roles of NO and the various
distinct members of the NOS family. (See, e.g., Marletta, J. Biol.
Chem. 268:12231-12234 (1993); Knowles et al., Biochem. J.
298:249-258 (1994)). It is well known by those skilled in the art
that multiple isoforms of the NOS enzyme exist and that they are
generally classified into two broad categories: 1) constitutive and
2) inducible. These classes of NOS enzymes vary considerably in
their size, amino acid sequence, activity and regulation, and
exhibit a number of substantial differences, indicating differences
in their molecular structures. Increasingly diverse biological
functions are being attributed to the NO formed by these three
major known types of NOS (Nathan, FASEB J. 6:3051 (1992); Marletta,
J. Biol. Chem. 268:12231 (1993); Knowles et al., Biochem. J.
298:249 (1994); Griffith et al., Annu. Rev. Physiol. 57:707
(1995)). For example, cells such as neurons and vascular
endothelial cells contain constitutive NOS isotypes, while
macrophages and vascular endothelial cells express an inducible
NOS.
[0006] Several isoforms of NOS's from different mammalian tissues
and cells have been purified and characterized; in brain (nNOS) by
Bredt and Synder, Proc. Natl. Acad. Sci. USA 87:682-685 (1990); in
endothelial cells (eNOS) by Forstermann et al., Biochem. Pharmacol.
42:1849-1857 (1991); in macrophages (iNOS) by Hibbs et al., Science
235:473 (1987) and Stuehr et al., Proc. Natl. Acad. Sci U. S. A.
88:7773-7777 (1991); in hepatocytes by Knowles et al., Biochem. J.
279:833-836 (1990); in vascular cells by Wood et al., Biochem.
Biophys. Res. Comm. 170:80-88; in neutrophils by Yui et al., J.
Biol. Chem. 266:12544-12547 (1991) and Yui et al., J. Biol. Chem.
266:3369-3371 (1991); and in other tissues (see, e.g., Hevel et
al., J. Biol. Chem. 266:22789-22791 (1991); Mayer et al., FEBS
Lett. 277:215-219 (1990); Schmidt et al., Proc. Natl. Acad. Sci U
S. A. 88:365-369 (1991); Ohshima et al., Biochem. Biophys. Res.
Commun. 183:238-244 (1992); Hiki et al., J. Biochem. 111:556-558
(1992); Evans et al., Proc. Natl. Acad. Sci U. S. A. 89:5361-5365
(1992); Sherman et al., Biochemistry 32:11600-11605 (1993)). U.S.
Pat. No. 5,268,465 claims a cDNA molecule encoding all or a portion
of a mammalian calmodulin-dependent nitric oxide synthase (nNOS),
comprising between 12 and 4,000 nucleotides. U.S. Pat. No.
5,468,630 claims an isolated nucleic acid molecule comprising the
nucleic acid sequence encoding a human inducible nitric oxide
synthase (iNOS) protein. U.S. Pat. No. 5,498,539 claims an isolated
nucleic acid molecule comprising the nucleic acid sequence encoding
a bovine endothelial nitric oxide synthase (eNOS) protein having
amino acid or nucleic acid sequences set forth in the
specification.
[0007] It is also known that small amounts of NO generated by a
constitutive NOS appear to act as a messenger molecule by
activating soluble guanylate cyclase and, thus, increasing
intracellular guanosine, 3', 5'-cyclic monophosphate (cGMP) and the
induction of biological responses that are dependent on cGMP as a
secondary messenger. For example, through this mechanism,
endothelial derived NO induces relaxation of vascular smooth muscle
and is identified as endothelium derived relaxing factor (Palmer et
al., Nature 327:524-526 (1987) and Ignarro et al., Proc. Natl.
Acad. Sci. USA 84:9265-9269 (1987)). In addition, neuronal nitric
oxide can act as a neuro-transmitter by activating guanylate
cyclase with important functions in the central nervous system and
autonomic nervous systems. Bredt and Synder, Proc. Natl. Acad. Sci.
USA 86:9030-9033 (1989) and Burnett et al., Science 257:401-403
(1992). Moreover, various purified NOS enzymes have been identified
as hemeproteins (Stuehr et al., J. Biol. Chem. 267:20547-20550
(1992); White et al., Biochemistry 31:6627-6631 (1992); McMillan et
al., Proc. Natl. Acad. Sci. USA 89:11141-11145 (1992)) and
flavoproteins (Hevel et al., J. Biol. Chem. 266:22789-22791 (1991);
Bredt et al., J. Biol. Chem. 267:10976-10981 (1992)).
[0008] Thus, the catalytic mechanisms of the NOS enzymes have also
been the subject of great interest (see, e.g., Marletta, J. Biol.
Chem. 268:12231-12234 (1993)). Stable isotope studies have shown
when that when L-arginine is the substrate for the enzyme, NO
derives from one of the two equivalent guanidino nitrogens on the
arginine moiety (Ignarro et al., 1987; Palmer et al., 1988), and
that di-oxygen is the source of the oxygen atoms incorporated into
citrulline and NO (Kwon et al., J. Biol. Chem. 265:13442-13445
(1990); Leone et al., J. Biol. Chem. 266:23790-23795 (1991)).
Moreover, N.sup.G-hydroxy-L-arginine has been demonstrated to be an
oxidative intermediate in the catalytic process (Stuehr et al., J.
Biol. Chem. 266:6259-6263 (1991); Wallace et al., Biochem. Biophys.
Res. Commun. 176:528-534 (1991); Pufahl et al., Biochemistry
31:6822-6828 (1992); Klatt et al., J. Biol. Chem. 268:14781-14787
(1993)).
[0009] Co-factors involved in the conversion of L-arginine to
L-citrulline and NO synthesis include tetrahydrobiopterin
(BH.sub.4), flavin adenine nucleotide (FAD) , the flavin-ribitol
phosphate part of FAD (FMN) and the reduced form of nicotinamide
adenine dinucleotide phosphate (NADPH). In addition, when the NOS
enzyme has been derived from brain or endothelial cells, calcium
and calmodulin are also required. (Bredt et al., Nature 351:714-718
(1991); Lamas et al., Proc. Natl. Acad. Sci. U. S. A. 89:6348-6352
(1992); Lyons et al., J. Biol. Chem. 267:6370-6374 (1992);
Lowenstein et al., Proc. Natl. Acad. Sci. U. S. A. 89:6711-6715
(1992); Xie et al., Science 256:225-228 (1992)). Calmodulin is a
well-known protein binder for Ca.sup.2+, ubiquitously found in
plant and animal cells. The Ca.sup.2+-calmodulin complex thus
formed is known to bind to various target proteins in the cell, and
thereby alter their activity.
[0010] Thus, it will be appreciated that nitric oxide has both
normal physiologic intracellular and extracellular regulatory
functions. However, excessive production of nitric oxide is
detrimental. For example, when vascular endothelial cells are
stimulated to express a NOS enzyme by a bacterial endotoxin, such
as for example bacterial lipopolysaccharide (LPS), and inflammatory
cytokines are elevated, the excess amounts of nitric oxides that
are produced contribute to the vascular collapse seen in sepsis.
Busse and Mulsch, FEBS Lett. 265:133-136 (1990). It is also known
that when vascular cells are stimulated to express a NOS enzyme by
inflammatory cytokines, the excess amounts of nitric oxides cause
massive dilation of blood vessels and sustained hypotension
commonly encountered in septic shock, and contribute to the
eventual vascular collapse seen in sepsis. Id. It is also known
that overproduction of nitric oxide in the lungs stimulated by
immune complexes directly damages the lung. Mulligan et al., J.
Immunol. 148:3086-3092 (1992). Induction of nitric oxide synthase
in pancreatic islets impairs insulin secretion and contributes to
the onset of juvenile diabetes. Corbett et al., J. Biol. Chem.
266:21351 (1991).
[0011] Thus, it will be appreciated that there is a great need in
the medical community for the ability to control and regulate
specific forms of NOS, particularly given its role in maintaining
normal blood pressure and the devastating effect of excess NO on
the cardiovascular, gastrointestinal and respiratory systems in
humans. Thus considerable research has been expended to discover
inhibitors and regulators of NOS activity. However, until the
present invention, researchers have limited their work almost
exclusively to the premise that L-arginine is the most
physiologically relevant substrate of NOS. The studies have
extended no further than the fact that dipeptides, such as Arg-Arg
and Arg-Phe are also oxidized by crude NOS preparations derived
from cultured endothelial cells and macrophages (Hecker et al.,
FEBS Lett. 294:221 (1991); Hecker et al., Proc. Natl. Acad. Sci.
USA 87:8612 (1990); Hecker et al., J. Cardiovasc. Pharmacol.
20:S139 (1992)). Thus, although there have been many publications
directed to analyses of arginine analogs or derivatives which
inhibit NOS activity by blocking the use of arginine as a substrate
for NO synthesis, or to the cofactors necessary for the conversion
of arginine, no one until the present inventors has considered
peptide, oligopeptide or protein substrates for the NOS enzyme.
Hence, given the expectation that natural or synthetic
arginine-rich peptide, oligopeptide or protein antagonists can
function as NOS inhibitors, the present invention will greatly
expand the number and types of inhibitors available for the
regulation and control of NO production in the body. In addition,
the use of a NOS to supply deficient individuals with NO-forming
capability will be greatly enhanced by the purification of the
nNOS-II class of enzymes. The present invention, therefore, will
also provide many new ways to study the biological mechanisms
involved in NO synthesis.
SUMMARY OF THE INVENTION
[0012] The present invention relates to a novel constitutive nitric
oxide synthase, referred to hereinafter as nNOS-II, that has been
purified from mammalian neural tissue, and which is capable of
utilizing arginine-rich peptides, polypeptides and proteins (e.g.,
bradykinin), as well as L-arginine, as substrates. Unlike
previously defined NOS isoforms, nNOS-II is unique in that it is
calmodulin-dependent with L-arginine as substrate, but
calmodulin-independent with an arginine-rich polypeptide, such as
bradykinin (BK), as substrate. When BK is the substrate, both the
N- and the C-terminal arginines of the oligopeptide are oxidized to
citrullines by nNOS-II. See Equation I. 1
[0013] Moreover, with BK as substrate, NOS activity is
competitively inhibited by N.sup.G-methyl-,
N.sup.G-nitro-L-arginines, and BK receptor-antagonists, including
oligopeptide BK receptor-antagonists.
[0014] More particularly, the present invention relates to a
substantially purified nNOS-II protein.
[0015] In another embodiment, the present invention relates to a
mammalian brain-derived nitric oxide synthase protein purified to
an activity at least 6,360-fold, said protein having a denatured
molecular mass as determined by sodium dodecyl sulfate
polyacrylamide gel electrophoresis under reducing conditions of
about 105 kD, and a native homodimeric molecular mass as determined
by gel filtration of about 230 kD, requiring NADPH, FAD, FMN,
Ca.sup.2+ and tetrahydrobiopterin cofactors for the production of
nitric oxide either from L-arginine, or an analog or derivative
thereof, or from an arginine-rich peptide, oligopeptide, or protein
substrate. As used herein, the term "peptide" is arbitrarily
defined as a peptide chain having a single peptide bond,
"oligopeptide" is a peptide chain having from 2 to 14 peptide
bonds, inclusive, and "protein" is a peptide chain having 15 or
more peptide bonds. "Polypeptide" is intended to be generic to
"oligopeptide" and "protein." "Arginine-rich" means that the
peptide, oligopeptide, or protein has at least one sterically
accessible arginine moiety.
[0016] In another embodiment, the present invention relates to a
method of regulating or controlling nitric oxide production in a
mammalian subject comprising administering to the mammal a nitric
oxide-regulating amount of a peptide, oligopeptide, or protein
inhibitor of nitric oxide synthase, preferably nNOS-II. As used
herein, "mammalian subject" is intended to include human
subjects.
[0017] In one embodiment, the present invention relates to a method
of reducing the rate of nitric oxide production in a mammalian
subject comprising administering to the mammal a nitric oxide
inhibiting amount of a peptide, oligopeptide or protein inhibitor
of nitric oxide synthase, preferably nNOS-II. "Reducing the rate"
is intended to include a reduction to zero, in which case the
reduction would be understood to include, not only a decrease in
the rate of NO production or of the quantity of NO produced, but
also a prevention of excess NO production.
[0018] In another embodiment, the present invention relates to a
method of enhancing the rate of nitric oxide production in a
mammalian subject comprising administering to the mammal a nitric
oxide enhancing amount of nitric oxide synthase, preferably
nNOS-II. "Enhancing the rate" is intended to include a stimulating,
inducing or causing production of NO in a NO-deficient or
NO-defective subject, in which case the enhancement would be
understood to include stimulation, induction or initialization of
NO production in such subjects or individuals, as well as
increasing the rate of NO production or the quantity of NO
produced.
[0019] In still another embodiment, the present invention relates
to a method of preventing or treating a nitric oxide-mediated
disease or condition in a mammalian subject comprising
administering to the subject in need of such prevention or
treatment a therapeutically effective amount of a peptide,
oligopeptide, or protein inhibitor of nitric oxide synthase,
preferably nNOS-II.
[0020] Additional objects, advantages and novel features of the
invention will be set forth in part in the description which
follows, and in part will become apparent to those skilled in the
art on examination of the following, or may be learned by practice
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 depicts a DEAE-agarose column elution profile for
both NOS enzymes from rat cerebellum. L-arginine (.largecircle.)
NOS activity (calmodulin dependent), and BK (.circle-solid.) NOS
activity (calmodulin independent). Protein absorbance at 280 nm
(.quadrature.) from rat cerebellum was used to monitor the eluting
process.
[0022] FIG. 2 depicts an 8%, silver-stained SDS-polyacrylamide gel
showing: Lane 1, cytosol (10 .mu.g protein); Lane 2, eluate from
first DEAE-column (10 .mu.g); Lane 3, NADPH eluate from
2',5'-ADP-agarose column (2 .mu.g protein); Lane 4, eluate from
second DEAE-agarose column containing purified enzyme (0.5 .mu.g
protein); Lane 5, control with sample buffer; Lane 6, molecular
weight markers (Bio-Rad) of myosin (208,000), b-galactosidase
(115,000) phosphorylase B (107,000), bovine serum albumin (79,500),
ovalbumin (49,500), carbonic anhydrase (34,800), soybean trypsin
(28,300).
[0023] FIG. 3 depicts HPLC chromatograms of nNOS-II reactions with
BK or Cit.sup.1-BK. Sensitivities of the regions at 20-30 minutes
are expanded 3-fold. FIG. 3A is a chromatogram of the enzymatic
reaction in which BK is the substrate. FIG. 3B is a chromatogram of
the enzymatic reaction in which Cit.sup.1-BK is the substrate.
[0024] FIG. 4 depicts double reciprocal plots of the inhibition of
NOS-II by
[N-adamantaneacetyl-D-Arg.sup.0,Hyp.sup.3,Thi.sup.5,8,D-Phe.sup.7]-BK
in the presence of BK. The inhibition experiments were carried out
the same as for standard enzyme assays under initial velocity
conditions. Concentrations of
[N-adamantaneacetyl-D-Arg.sup.0,Hyp.sup.3,Thi.sup.5,8,D-
-Phe.sup.7]-BK were closed circles, 0 .mu.M; open circles, 0.625
.mu.M; closed squares, 1.25 .mu.M; open squares, 2.5 .mu.M; closed
triangles, 5 .mu.M; open triangles, 10 .mu.M. The values are the
mean of three measurements.
[0025] FIG. 5 depicts DEAE-agarose column elution profile for NOSs
and protein (absorbance at 280 nm.) from rat cerebellum. L-arginine
(O) NOS activity (calmodulin dependent) and BK (.circle-solid.) NOS
activity (calmodulin independent).
[0026] FIG. 6 depicts an 8%, silver-stained SDS-polyacrylamide gel
showing: Lane 1, cytosol (10 .mu.g protein); Lane 2, eluate from
first DEAE-agarose column (10 .mu.g); Lane 3, NADPH eluate from
2'-5'-ADP-agarose column (2 .mu.g protein); Lane 4, eluate from
second DEAE-agarose column containing purified enzyme (0.5 .mu.g
protein); Lane 5, control with sample buffer; Lane 6, molecular
weight markers (Bio-Rad) of myosin (208,000). b-galactosidase
(115,000) phosphorylase B (107,000), bovine serum albumin (79,500),
ovalbumin (49,500), carbonic anhydrase (34,800), soybean trypsin
(28,300).
[0027] FIG. 7 depicts HPLC chromatograms of nNOS-II reactions with
BK or Cit.sup.1-BK. Sensitivies of the regions at 20-30 minutes are
expanded 3-fold. (A) Chromatogram of enzymatic reaction with BK as
substrate. (B) Chromatogram of enzymatic reaction with Cit.sup.1-BK
as substrate.
[0028] FIG. 8 depicts the oxidation of bradykinin (BK) to
Cit.sup.1.Cit.sup.9-BK and nitric oxide (NO) by nNOS-II.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0029] The present invention is directed to a novel constitutive
mammalian nitric oxide synthase (NOS) that utilizes arginine-rich
peptides, oligopeptides (e.g., bradykinin), and proteins, as well
as L-arginine, as substrates. The present invention was begun
following an earlier discovery by the inventors of a novel
bacterial NOS that utilized a range of arginine-containing
polypeptides, including BK and related BK analogs ranging from
6-mer to 11-mer, to produce nitric oxide. (See, Chen and Rosazza,
Biochem. Biophys. Res. Commun. 203:1251 (1994) and Chen and
Rosazza, J. Bacteriol. 177:5122 (1995)).
[0030] Although increasingly diverse biological functions are being
attributed to NO formed by three major known types of NOS (Nathan,
FASEB J. 6:3051-3064 (1992); Marletta, J. Biol. Chem.
268:12231-12234 (1993); Knowles et al., Biochem. J. 298:249-258
(1994); Griffith et al., Annu. Rev. Physiol. 57:707-736 (1995)),
there are no known published reports of arginine-containing
oligopeptides, polypeptides or proteins acting as a substrate for
any NOS enzyme. To the contrary, throughout the prior art,
L-arginine is currently recognized as the only physiologically
relevant NOS-substrate.
[0031] The present inventors found that upon purification of crude
supernatant preparations from rat cerebellum using weak anion
exchange chromatography (DEAE-agarose), the elution profile
reproducibly produces two calmodulin-dependent neuronal nitric
oxide synthase (nNOS) peaks with L-arginine as substrate. Both
peaks display the characteristic calmodulin-dependent nNOS activity
using L-arginine as substrate, as described by Bredt et al., PNAS
87:682(1990). However, the novel enzyme of the second peak
(fractions 15-21), which has not been studied in the prior art,
further displays unique calmodulin-independent nNOS activity in the
presence of an arginine-rich polypeptide substrate, such as BK.
(NOS is measured by the conversion of oxyhemoglobin to
methemoglobin.) This novel nitric oxide synthase has been purified
to afford a 6,360-fold purified enzyme preparation, and designated
nNOS-II. When a species of nNOS-II acts upon a particular
substrate, such as BK, the enzyme name may further denominate the
substrate, e.g., nNOS.sub.BK. It should be emphasized, however,
that BK is only one of many peptide, oligopeptide or protein
substrates for nNOS-II.
[0032] Purified nNOS-II has a Mr of 105 kD by SDS-PAGE analysis and
an apparent native M.sub.r of 230 kD by gel filtration, indicating
that the enzyme is a homodimeric protein. By comparison, the
previously described, purified nNOS enzyme migrates as a single 160
kD band on SDS-PAGE, and the native enzyme appears to be a monomer.
However, both enzymes require the presence of NAPDH, FAD, FMN,
Ca.sup.2+, and tetrahydrobiopterin cofactors for substrate
oxidation to occur.
[0033] When calmodulin is present with the necessary cofactors,
nNOS-II also oxidizes L-arginine, but at a K.sub.M of 10.6 .mu.M,
slightly higher than the previously reported K.sub.M of 2 .mu.M for
nNOS. (K.sub.M is the substrate concentration that allows the
reaction to proceed at one-half its maximum rate.) Moreover, with
an L-arginine substrate, the rate of reaction, V.sub.max, value
displayed by nNOS-II is 0.85 .mu.mol/min/mg protein, while that
reported for the nNOS of the prior art is 0.96 .mu.mol/min/mg
protein. (V.sub.max refers to the rate of the enzyme reaction,
depending only upon how rapidly the substrate molecule can be
processed. This rate when divided by the enzyme concentration
provides the turnover number.)
[0034] Finally, numerous reports describe the abolishment of nNOS
activity (as defined by Bredt and Snyder) by the addition of
quantities of L-arginine analog inhibitors. However, in marked
contrast, with nNOS-II using BK as substrate,
N.sup.G-methyl-L-arginine(L-NMA) and
N.sup.G-nitro-L-arginine(L-NNA) were found to be reversible
competitive inhibitors with apparent Ki values of 8.6 .mu.M and
23.8 .mu.M, respectively. These values are significantly different
for the reported Ki values for L-NMA of 1.4 .mu.M and for L-NNA of
4.4 .mu.M for nNOS with L-arginine as substrate.
[0035] Thus, the present invention comprises a novel isoform of
nitric oxide synthase, herein designated as nNOS-II, which can be
readily distinguished from all previously reported NOS species,
including nNOS. The broader implications of this discovery include
the future identification of a new class of native, recombinant, or
synthetic peptides that will function as NOS inhibitors for the
modulation of cardiovascular, gastrointestinal, or bronchial
activities, for contraceptive control, for the management of opioid
withdrawal or cocaine-induced toxicity, or for the prevention or
treatment of certain nitric oxide-mediated pathogenic conditions,
such as ischemic stroke, diabetes, systemic hypotension, multiple
sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's
disease, and the like.
[0036] It will be understood by those skilled in the art that the
present invention is not limited to the use of any specific
peptide, oligopeptide or protein as the substrate for the nNOS-II
although preferred substrates will be arginine-rich peptides,
oligopeptides or proteins. More preferrably, the tertiary structure
of such a peptide, oligopeptide or protein substrate will be such
that one or more arginine groups are available to the NOS enzyme.
It is most preferred that the substrate be an oligopeptide of
managable size, such as from 6 to 11 amino acid residues, wherein
both the .alpha. and .omega. amino acids are arginine.
[0037] Exemplary peptides, oligopeptides and proteins upon which
nNOS-II have been shown to be active are set forth in Table 1:
1TABLE 1 Arginine-Containing Peptides or Oligopeptides as
Substrates for nNOS-II.sup.a Relative Activity Peptide Amino Acid
Sequence (%) L-arginine N/A.sup.b 100.sup.c Poly-arginine N/A 30
(M.sub.r 5,000) BK Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 125 (SEQ ID
NO: 1) Des-Arg.sup.1-BK Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 94 (SEQ ID
NO: 2) Des-Arg.sup.9-BK Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe 180 (SEQ ID
NO: 3) BK fragment 1-7 Arg-Pro-Pro-Gly-Phe-Ser-Pro 80 (SEQ ID NO:
4) BK fragment 1-5 Arg-Pro-Pro-Gly-Phe (SEQ ID NO: 5) 61 BK
fragment 2-7 Pro-Pro-Gly-Phe-Ser-Pro (SEQ ID NO: 6) 0
[Lys.sup.1]-BK Lys-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 113 (SEQ ID NO:
7) Lys-BK Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 116 (SEQ ID NO:
8) Ile-Ser-BK Ile-Ser-Arg-Pro-Pro-Gly-Phe-Ser-- Pro-Phe- 110 Arg
(SEQ ID NO: 9) Met-Lys-BK Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-
0 Arg (SEQ ID NO: 10) .sup.aStandard enzymatic assay was carried
out to measure initial velocity of the enzymatic reaction. Purified
nNOS-II 0.2 .mu.g (specific activity for BK, 280 nmole NO
formed/min/mg protein) was used for the assays. Substrate
concentrations were 100 .mu.M except for poly-arginine used with 5
.mu.g/ml. .sup.bNot applicable. .sup.cActivity for arginine is
expressed as 100%. The values are the means of three
measurements.
[0038] This list, however, is intended to be merely exemplary, not
limiting in scope.
[0039] A model substrate of nNOS-II is the nonapeptide bradykinin
(Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) (SEQ ID NO:1). BK was
selected as a convenient and available model oligopeptide substrate
for displaying the catalytic properties of nNOS-II because:
[0040] it contains positionally different N- and C-terminal
arginine residues;
[0041] its recognized physiological role in both cardiovascular and
central nervous systems has been well characterized;
[0042] it displays physiological and pharmacological effects
similar to those ascribed to NO; and
[0043] it has been linked as an apparent mediator of NOS activity
(Regoli et al., Pharmacol. Rev. 32:1-46 (1980); Bhoola, Pharmacol.
Rev. 44:1-58 (1992)). In addition, BK is of ideal size (containing
9 amino acid residues) for conversion by the nNOS-II enzyme.
[0044] In a preferred embodiment, the present invention provides
substantially pure nNOS-II protein. One specific enzyme herein
provided in purified form is nNOS-II. When used with regard to
nNOS-II of the present invention, the terms "protein" and "enzyme"
are used interchangeably, even though technically an enzyme is a
specific subset of the category "protein." Moreover, as used
herein, a protein is said to be "highly purified" or "substantially
pure" if the specific activity of the protein cannot be
significantly incresed by further purification, and if the specific
activity is greater than that found in whole cell extracts
containing the protein.
[0045] Any eukaryotic organism can be used as a source of nNOS-II
or the genes encoding same, as long as the source organism
naturally contains the enzyme or its equivalent. As used herein,
"source organism" refers to the original organism from which the
amino acid or DNA sequence is derived, regardless of the organism
the enzyme expressed in or ultimately isolated from. For example, a
human is said to be the "source organism" of nNOS-II expressed by a
bacterial expression system as long as the amino acid sequence is
that of human nNOS-II. The most preferred source organism is
mammalian.
[0046] A variety of methodologies known in the art can be utilized
to obtain the nNOS-II proteins of the present invention. In one
embodiment, the enzyme is purified from tissues or cells which
naturally produce it, such as rat cerebellum. One skilled in the
art can readily follow known methods for isolating proteins in
order to obtain the nNOS-II proteins. These include, but are not
limited to, immunochromotography, size-exclusion chromatography,
ion-exchange chromatography, affinity chromatography, HPLC, and the
methods set forth by example in the present disclosure. One skilled
in the art can readily adapt known purification schemes to delete
certain steps or to incorporate additional purification
procedures.
[0047] In a preferred embodiment of the invention, nNOS-II may be
purified using column chromatography. Specifically, it has been
found that greater than one-thousand-fold purification can be
achieved using an affinity chromatography column. Since NADPH is a
necessary cofactor for enzyme activity, if one employs a solid
matrix containing an NADPH moiety or an NADPH analog, such as
dextran blue, or 2',5'-ADP agarose or 2',5'-ADP sepharose, then the
NOS of the present invention will bind to the matrix. It can be
eluted using a soluble form of NADPH or an analog thereof at a
concentration of about 1 to about 10 mM. It is desirable that the
preparation which is applied to the affinity chromatography column
first be partially purified on an ion exchange column, such as,
diethylaminoethyl (DEAE) agarose. Other ion exchange columns known
in the art can also be used. The NOS of the present invention binds
to DEAE-agarose and can be eluted with a sodium chloride gradient.
The greatest peak of nNOS-II activity elutes with between about 150
mM and about 210 mM sodium chloride. A combination of these three
column chromatography processes on a cleared brain homogenate will
result in a homogeneous preparation, as can be demonstrated by
silver staining of an SDS/PAGE-separated sample, or by Western
blotting.
[0048] In another embodiment, the enzyme may be purified from cells
which have been altered to express the desired protein. As used
herein, a cell is said to be "altered to express a desired protein"
when the cell, through genetic manipulation, is made to produce a
protein which it normally does not produce, or which the cell
normally produces at low levels. One skilled in the art can readily
adapt procedures for introducing and expressing either genomic or
cDNA sequences into either eukaryotic or prokaryotic cells, in
order to generate a cell which produces the desired nNOS-II
protein.
[0049] The present invention further encompasses the expression of
the nNOS-II proteins (or a functional derivative thereof) in either
prokaryotic or eukaryotic cells. A "functional derivative" of a
sequence, either protein or nucleic acid, is a molecule that
possesses a biological activity (either functional or structural)
that is substantially similar to a biological activity of the
protein or nucleic acid sequence. A functional derivative of a
protein may or may not contain post-translational modifications
such as covalently linked carbohydrate, depending on the necessity
of such modifications for the performance of a specific function.
The term "functional derivative" is intended to include the
"fragments," "segments," "variants," "analogs," or "chemical
derivatives" of a molecule.
[0050] As used herein, a molecule is said to be a "chemical
derivative" of another molecule when it contains additional
chemical moieties not normally a part of the molecule. Such
moieties may improve the molecule's solubility, absorption,
biological half life, and the like. The moieties may alternatively
decrease the toxicity of the molecule, eliminate or attenuate any
undesirable side effect of the molecule, and the like. Moieties
capable of mediating such effects are disclosed in Remington's
Pharmaceutical Sciences (1980). Procedures for coupling such
moieties to a molecule are well known in the art.
[0051] A "variant" or "allelic or species variant" of a protein or
nucleic acid is meant to refer to a molecule substantially similar
in structure and biological activity to either the protein or
nucleic acid. Thus, provided that two molecules possess a common
activity and may substitute for each other, they are considered
variants as that term is used herein even if the composition or
secondary, tertiary, or quaternary structure of one of the
molecules is not identical to that found in the other, or if the
amino acid or nucleotide sequence is not identical.
[0052] Preferred prokaryotic hosts include bacteria such as E.
coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia,
etc. Under such conditions, the nNOS-II will not be glycosylated.
The prokaryotic host must be compatible with the replicon and
control sequences in the expression plasmid.
[0053] However, prokaryotic systems may not prove efficacious for
the expression of all proteins. While prokaryotic expression
systems, e.g., pET3c, have been used to express high molecular
weight proteins, such as a biologically active (molecular weight
(M.sub.r).about.118 kDa) FGF-1:.beta.-galactosidase chimera (Shi et
al., submitted to J. Biol. Chem., 1996), successful folding and
disulfide bond formation may be difficult to accomplish in
bacteria.
[0054] Nevertheless, to express nNOS-II (or a functional derivative
thereof) in a prokaryotic cell, it is necessary to operably link
the nNOS-II coding sequence to a functional prokaryotic promoter.
Such promoters may be either constitutive or, more preferably,
regulatable (i.e., inducible or derepressible). Examples of
constitutive promoters include the int promoter of bacteriophage
.lambda., the bla promoter of the .beta.-lactamase gene sequence of
pBR322, and the CAT promoter of the chloramphenicol acetyl
transferase gene sequence of pPR325, etc. Examples of inducible
prokaryotic promoters include the major right and left promoters of
bacteriophage .lambda. (P.sub.L and P.sub.R), the trp, recA, lacZ,
lacI, and gal promoters of E. coli, the .alpha.-amylase (Ulmanen et
al., J. Bacteriol. 162:176-182 (1985)) and the .zeta.-28-specific
promoters of B. subtilis (Gilman et al., Gene sequence 32:11-20
(1984)), the promoters of the bacteriophages of Bacillus (Gryczan,
In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY
(1982)), and Streptomyces promoters (Ward et al., Mol. Gen. Genet.
203:468-478 (1986)). See also reviews by Glick (J. Ind. Microbiol.
1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); and
Gottesman (Ann. Rev. Genet. 18:415-442 (1984)).
[0055] Proper expression in a prokaryotic cell also requires the
presence of a ribosome binding site upstream of the gene
sequence-encoding sequence. Such ribosome binding sites are
disclosed, for example, by Gold et al. (Ann. Rev. Microbiol.
35:365-404 (1981)).
[0056] Preferred eukaryotic hosts include yeast, fungi, insect
cells, mammalian cells, either in vivo or in tissue culture.
Mammalian cells which may be useful as hosts include HeLa cells,
cells of fibroblast origin such as VERO or CHO-K1, or cells of
lymphoid origin, such as the hybridoma SP2/O-AG14 or the myeloma
P3.times.63Sg8, and their derivatives. Preferred mammalian host
cells include SP2/0 and J558L, as well as neuroblastoma cell lines
such as IMR 332 that may provide better capacities for correct
post-translational processing.
[0057] For a mammalian host, several possible vector systems are
available for the expression of nNOS-II. A wide variety of
transcriptional and translational regulatory sequences may be
employed, depending upon the nature of the host. The
transcriptional and translational regulatory signals may be derived
from viral sources, such as adenovirus, bovine papilloma virus,
Simian virus, or the like, where the regulatory signals are
associated with a particular gene sequence which has a high level
of expression. Alternatively, promoters from mammalian expression
products, such as actin, collagen, myosin, etc., may be employed.
Transcriptional initiation regulatory signals may be selected which
allow for repression or activation, so that expression of the gene
sequences can be modulated. Of interest are regulatory signals
which are temperature-sensitive so that by varying the temperature,
expression can be repressed or initiated, or are subject to
chemical (such as metabolite) regulation.
[0058] Yeast expression systems can also carry out
post-translational peptide modifications. A number of recombinant
DNA strategies exist which utilize strong promoter sequences and
high copy number of plasmids which can be utilized for production
of the desired proteins in yeast. Yeast recognizes leader sequences
on cloned mammalian gene sequence products and secretes peptides
bearing leader sequences (i.e., pre-peptides). Any of a series of
yeast gene sequence expression systems incorporating promoter and
termination elements from the actively expressed gene sequences
coding for glycolytic enzymes produced in large quantities when
yeast are grown in mediums rich in glucose can be utilized. Known
glycolytic gene sequences can also provide very efficient
transcriptional control signals. For example, the promoter and
terminator signals of the phosphoglycerate kinase gene sequence can
be utilized.
[0059] The more preferred host for a protein the size of nNOS-II is
insect cells, for example the Drosophila larvae. Using insect cells
as hosts, the Drosophila alcohol dehydrogenase promoter can be used
(see, e.g., Rubin, G. M., Science 240:1453-1459 (1988)).
[0060] The baculovirus insect cell expression system is the most
preferred system for expressing the soluble nNOS-II construct as a
carboxy-terminal triple tandem myc-epitope repeat:
glutathione-S-transferase (GST) fusion protein chimera, using
conventional PCR methods (Zhan et al., J. Biol. Chem.
269:20221-20224 (1994)). These include the use of recombinant
circle PCR to synthesize the soluble nNOS-II construct, the
preparation and expression of the recombinant virus, AcNPV-GsJ in
Sf9 cells (Summers and Smith (1988) A Manual of Methods for
Baculovirus Vectors and Insect Culture Procedures (Texas
Experimental Station Bulletin #1555)), the use of GST affinity
chromatography (Zhan et al., 1994) and reversed phase or ion
exchange HPLC to purify the recombinant protein from Sf9 cell
lysates and Myc immunoblot analysis to monitor the purification and
assess the purity of the nNOS-II protein.
[0061] The soluble nNOS-II construct may not only prove to be
valuable for the baculovirus expression system, but also as a
construct for the expression of a secreted and soluble nNOS-II
enzyme in mammalian cells for implantation in vivo. Moreover,
baculovirus vectors can be engineered to express large amounts of
nNOS-II in insect cells (Jasny, Science 238:1653 (1987); Miller et
al., in Genetic Engineering (1986), Setlow et al., eds., Plenum,
Vol. 8, pp. 277-297).
[0062] As discussed above, expression of nNOS-II in eukaryotic
hosts requires the use of eukaryotic regulatory regions. Such
regions will, in general, include a promoter region sufficient to
direct the initiation of RNA synthesis. Preferred eukaryotic
promoters include: the promoter of the mouse metallothionein I gene
sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the
TK promoter of Herpes virus (McKnight, Cell 31:355-365 (1982)); the
SV40 early promoter (Benoist et al., Nature (London) 290:304-310
(1981)); the yeast gal4 gene sequence promoter (Johnston et al.,
Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver et al.,
Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).
[0063] As is widely known, translation of eukaryotic mRNA is
initiated at the codon which encodes the first methionine. For this
reason, it is preferable to ensure that the linkage between a
eukaryotic promoter and a DNA sequence which encodes nNOS-II (or a
functional derivative thereof) does not contain any intervening
codons which are capable of encoding a methionine (i.e., AUG). The
presence of such codons results either in a formation of a fusion
protein (if the AUG codon is in the same reading frame as the
nNOS-II coding sequence) or a frame-shift a frame-shift mutation
(if the AUG codon is not in the same reading frame as the nNOS-II
coding sequence).
[0064] The nNOS-II coding sequence and an operably linked promoter
may be introduced into a recipient prokaryotic or eukaryotic cell
either as a non-replicating DNA (or RNA) molecule, which may either
be a linear molecule or, more preferably, a closed covalent
circular molecule. Since such molecules are incapable of autonomous
replication, the expression of the nNOS-II may occur through the
transient expression of the introduced sequence. Alternatively,
permanent expression may occur through the integration of the
introduced sequence into the host chromosome.
[0065] In one embodiment, a vector is employed which is capable of
integrating the desired gene sequences into the host cell
chromosome. Cells which have stably integrated the introduced DNA
into their chromosomes can be selected by also introducing one or
more markers which allow for selection of host cells which contain
the expression vector. The marker may provide for prototrophy to an
auxotrophic host, biocide resistance, e.g., antibiotics, or heavy
metals, such as copper, or the like. The selectable marker gene
sequence can either be directly linked to the DNA gene sequences to
be expressed, or introduced into the same cell by co-transfection.
Additional elements may also be needed for optimal synthesis of
single chain binding protein mRNA. These elements may include
splice signals, as well as transcription promoters, enhancers, and
termination signals. cDNA expression vectors incorporating such
elements include those described by Okayama, H., Molec. Cell. Biol.
3:280 (1983).
[0066] In a preferred embodiment, the introduced sequence will be
incorporated into a plasmid or viral vector capable of autonomous
replication in the recipient host. Any of a wide variety of vectors
may be employed for this purpose. Factors of importance in
selecting a particular plasmid or viral vector include: the ease
with which recipient cells that contain the vector may be
recognized and selected from those recipient cells which do not
contain the vector; the number of copies of the vector which are
desired in a particular host; and whether it is desirable to be
able to "shuttle" the vector between host cells of different
species.
[0067] Preferred prokaryotic vectors include plasmids, such as
those capable of replication in E. coli (such as, for example,
pBR322, ColE1, pSC101, pACYC 184, .pi.VX). Such plasmids are, for
example, disclosed by Maniatis et al. (In: Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1982)). Bacillus plasmids include pC194, pC221, pT127, etc.
Such plasmids are disclosed by Gryczan (In: The Molecular Biology
of the Bacilli, Academic Press, N.Y. (1982), pp. 307-329). Suitable
Streptomyces plasmids include pIJ101 (Kendall et al., J. Bacteriol.
169:4177-4183 (1987)), and streptomyces bacteriophages such as
.phi.C31 (Chater et al., In: Sixth International Symposium on
Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986),
pp. 45-54). Pseudomonas plasmids are reviewed by John et al. (Rev.
Infect. Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol.
33:729-742 (1978)).
[0068] Preferred eukaryotic plasmids include BPV, vaccinia, SV40,
2-micron circle, etc., or their derivatives. Such plasmids are well
known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274
(1982); Broach In: The Molecular Biology of the Yeast
Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach,
Cell 28:203-204 (1982); Bollon et al., J. Clin. Hematol. Oncol.
10:39-48 (1980); Maniatis, In: Cell Biology: A Comprehensive
Treatise, Vol. 3, Gene sequence Expression, Academic Press, NY, pp.
563-608 (1980)).
[0069] Once the vector or DNA sequence containing the construct(s)
has been prepared for expression, the DNA construct(s) may be
introduced into an appropriate host cell by any of a variety of
suitable means: transformation, transfection, conjugation,
protoplast fusion, electroporation, calcium
phosphate-precipitation, direct microinjection, etc. After the
introduction of the vector, recipient cells are grown in a
selective medium, which selects for the growth of vector-containing
cells. Expression of the cloned gene sequence(s) results in the
production of nNOS-II, or fragments thereof. This can take place in
the transformed cells as such, or following the induction of these
cells to differentiate (for example, by administration of
bromodeoxyuracil to neuroblastoma cells or the like).
[0070] The nNOS-II proteins (or a functional derivatives thereof)
of the present invention can be used in a variety of procedures and
methods, such as for the generation of antibodies, for use in
identifying pharmaceutical compositions, for studying DNA/protein
interaction, and for examinining the mechanism of NO synthesis.
[0071] The substantially pure peptides of the present invention may
also be administered to a mammal intravenously, intramuscularly,
subcutaneously, enterally, topically or parenterally. A
"substantially pure" or "highly purified" protein, as defined
previously, is a protein preparation that is generally lacking in
other cellular components with which it is normally associated in
vivo.
[0072] When administering peptides by injection, the administration
may be by continuous injections, or by single or multiple
injections. The peptides are intended to be provided to a recipient
mammal in a "pharmacologically or pharmaceutically acceptable form"
in an amount sufficient to "therapeutically effective." A peptide
is considered to be in "pharmaceutically or pharmacologically
acceptable form" if its administration can be tolerated by a
recipient patient. An amount is said to be "therapeutically
effective" (an "effective amount") if the dosage, route of
administration, etc., of the agent are sufficient to effect a
response to nNOS-II. Thus, the present peptides may be used to
induce, increase, enhance, control, regulate or modulate the effect
of the nNOS-II protein, or the synthesis and expression of NO.
[0073] In another embodiment of the present invention, methods for
inhibiting, decreasing or preventing the activity of the nNOS-II
protein can be achieved by providing an agent capable of binding to
or inhibiting the enzyme (or a functional derivative thereof). Such
agents include, but are not limited to: nNOS-II inhibitors and
antagonists, antisense nNOS-II, the antibodies to nNOS-II
(anti-nNOS-II), and the secondary or anti-peptide peptides of the
present invention. By decreasing the activity of nNOS-II the effect
which expression of the peptide has on NO synthesis can be
modified, regulated, controlled, inhibited or prevented.
[0074] In one example of the present invention, methods are
presented for decreasing the activity of nNOS-II (or a functional
derivative thereof) by means of an anti-sense strand of cDNA to
disrupt the translation of the nNOS-II message. Specifically, a
cell is modified using routine procedures such that it expresses an
antisense message, a message which is complementary to the
pseudogene message. By constitutively or inducibly expressing the
antisense RNA, the translation of the nNOS-II mRNA can be
regulated. Such antisense technology has been successfully applied
to regulate the expression of poly(ADP-ribose) polymerase (see,
Ding et al., J. Biol. Chem. 267 (1992)).
[0075] In the alternative, nNOS-II activity can be prevented or
inhibited by binding the peptide, oligopeptide or protein substrate
used by the enzyme, thus modifying the amount of NO can be
synthesized by the enzyme. Examples of such nNOS inhibitors include
peptides or petidomimetics of structures such as bradykinin B2
receptor antagonists that have demonstrated nNOS-II inhibition
activity, or arginine derivatives, such as L-NNA or L-NMA. Not all
bradykinin B2 receptor antagonists have nNOS-II inhibitory activity
as demonstrated in the present invention; however, it is within the
ordinary skill of one in the art using known techniques and the
procedures herein disclosed to determine which peptides,
oligopeptides or proteins are capable of nNOS-II inhibition.
[0076] On the other hand, methods for stimulating, increasing or
enhancing the activity of the nNOS-II peptide can be achieved by
providing an agent capable of modulating the synthesis of NO by
nNOS-II (or a functional derivative thereof), or by inhibiting or
preventing an inhibitory signal which would diminish or stop the
activity of nNOS-II in the system. Such agents include, but are not
limited to, the anti-antisense nNOS-II peptides. By enhancing the
activity of nNOS-II the effect which the enzyme has on NO synthesis
can also be modified.
[0077] In yet another embodiment, nNOS-II (or a functional
derivative or variant thereof) can be used to produce antibodies or
hybridomas. One skilled in the art will recognize that if an
antibody is desired that will bind to nNOS-II such an antibody
would be generated as described above and used as an immunogen. The
resulting antibodies are screened for the ability to bind
nNOS-II.
[0078] The antibodies utilized in the above methods can be
monoclonal or polyclonal antibodies, as well fragments of these
antibodies and humanized forms. Humanized forms of the antibodies
of the present invention may be generated using one of the
procedures known in the art such as chimerization or CDR
grafting.
[0079] In general, techniques for preparing monoclonal antibodies
are well known in the art (Campbell, "Monoclonal Antibody
Technology: Laboratory Techniques in Biochemistry and Molecular
Biology," Elsevier Science Publishers, Amsterdam, The Netherlands
(1984); St. Groth et al., J. Immunol. Methods 35:1-21 (1980). For
example, in one embodiment an antibody capable of binding nNOS-II
is generated by immunizing an animal with a synthetic polypeptide
whose sequence is obtained from a region of the nNOS-II
protein.
[0080] Any animal (mouse, rabbit, etc.) which is known to produce
antibodies can be utilized to produce antibodies with the desired
specificity, although because of the large size of the nNOS-II
molecule, the rabbit may be preferred. Methods for immunization are
well known in the art. Such methods include subcutaneous or
interperitoneal injection of the polypeptide. One skilled in the
art will recognize that the amount of polypeptide used for
immunization will vary based on the animal which is immunized, the
antigenicity of the polypeptide and the site of injection.
[0081] The polypeptide may be modified or administered in an
adjuvant in order to increase the peptide antigenicity. Methods of
increasing the antigenicity of a polypeptide are well known in the
art. Such procedures include coupling the antigen with a
heterologous protein (such as globulin or .beta.-galactosidase) or
through the inclusion of an adjuvant during immunization.
[0082] For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, such as SP2/0-Ag14
myeloma cells, and allowed to become monoclonal antibody producing
hybridoma cells. A hybridoma is an immortalized cell line which is
capable of secreting a specific monoclonal antibody.
[0083] Any one of a number of methods well known in the art can be
used to identify the hybridoma cell which produces an antibody with
the desired characteristics. These include screening the hybridomas
with an ELISA assay, western blot analysis, or radioimmunoassay
(Lutz et al., Exp. Cell Res. 175:109-124 (1988)).
[0084] Hybridomas secreting the desired antibodies are cloned and
the class and subclass are determined using procedures known in the
art (Campbell, Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1984)).
[0085] For polyclonal antibodies, antibody containing antisera is
isolated from the immunized animal and is screened for the presence
of antibodies with the desired specificity using one of the
above-described procedures.
[0086] Conditions for incubating an antibody with a test sample
vary. Incubating conditions depend on the format employed in the
assay, the detection methods employed the nature of the test
sample, and the type and nature of the antibody used in the assay.
One skilled in the art will recognize that any one of the commonly
available immunological assay formats (such as, radioimmunoassays,
enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or
rocket immunofluorescent assays, or the like) can readily be
adapted to employ the antibodies of the present invention. Examples
of such assays can be found in Chard, "An Introduction to
Radioimmunoassay and Related Techniques" Elsevier Science
Publishers, Amsterdam, The Netherlands (1986); Bullock et al.,
"Techniques in Immunocytochemistry," Academic Press, Orlando, Fla.
Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, "Practice and
Theory of Enzyme Immunoassays: Laboratory Techniques in
Biochemistry and Molecular Biology," Elsevier Science Publishers,
Amsterdam, The Netherlands (1985).
[0087] The anti-nNOS-II antibody and nNOS-inhibitors are also
effective when immobilized on a solid support. Examples of such
solid supports include, but are not limited to, plastics such as
polycarbonate, complex carbohydrates such as agarose and sepharose,
and acrylic resins, such as polyacrylamide and latex beads.
Techniques for coupling antibodies to such solid supports are well
known in the art (Weir et al., "Handbook of Experimental
Immunology" 4th Ed., Blackwell Scientific Publications, Oxford,
England, Chapter 10 (1986), Jacoby et al., Meth. Enzym. 34 Academic
Press, N.Y. (1974).
[0088] Additionally, one or more of the antibodies used in the
above described methods can be detectably labelled prior to use.
Antibodies can be detectably labelled through the use of
radioisotopes, affinity labels (such as, biotin, avidin, etc.),
enzymatic labels (such as, horse radish peroxidase, alkaline
phosphatase, etc.) fluorescent labels (such as, FITC or rhodamine,
etc.), paramagnetic atoms, etc. Procedures for accomplishing such
labelling are well-known in the art, for example, see Sternberger
et al., J. Histochem. Cytochem. 18:315 (1970), Bayer et al., Meth.
Enzym. 62:308 (1979), Engval et al., Immunol. 109:129 (1972),
Goding, J. Immunol. Meth. 13:215 (1976). The labeled antibodies of
the present invention can be used for in vitro, in vivo, and in
situ assays to identify cells or tissues which express a specific
protein or enzyme.
[0089] In an embodiment of the above methods, the antibodies are
labeled, such that a signal is produced when the antibody(s) bind
to the same molecule. One such system is described in U.S. Pat. No.
4,663,278.
[0090] The antibodies or antisense peptides of the present
invention may be administered to a mammal intravenously,
intramuscularly, subcutaneously, enterally, topically or
parenterally. When administering antibodies or peptides by
injection, the administration may be by continuous injections, or
by single or multiple injections.
[0091] The antibodies or antisense peptides of the present
invention are intended to be provided to a recipient mammal in a
"pharmaceutically acceptable form" in an amount sufficient to be
"therapeutically effective" or an "effective amount". As above, an
amount is said to be therapeutically effective (an effective
amount), if the dosage, route of administration, etc. of the agent
are sufficient to affect the response to nNOS-II. Thus, the present
antibodies may either stimulate or enhance the activity of the
nNOS-II protein, resulting in increased NO synthsis, or they may
inhibit or prevent the nNOS-II conversion of the peptide,
oligopeptide or protein substrate into NO. Or, secondary
antibody(s) may be designed to affect the response to the nNOS-II
per se, i.e., an anti-antibody to nNOS-II. In the alternative,
either an antibody or an anti-antibody may be designed to affect
only the anti-sense strand of the molecule.
[0092] One skilled in the art can readily adapt currently available
procedures to generate secondary antibody peptides capable of
binding to a specific peptide sequence in order to generate
rationally designed antipeptide peptides, for example see Hurby et
al., "Application of Synthetic Peptides: Antisense Peptides", In
Synthetic Peptides, A User's Guide, Freeman, N.Y., pp. 289-307
(1992), and Kaspczak et al., Biochemistry 28:9230-8 (1989). As used
herein, an agent is said to be "rationally selected or designed"
when the agent is chosen based on the configuration of the nNOS-II
peptide.
[0093] To detect secondary antibodies, or in the alternative, the
labelled primary antibody, labelling reagents may include, e.g.,
chromophobic, enzymatic, or antibody binding reagents which are
capable of reacting with the labelled antibody. One skilled in the
art will readily recognize that the disclosed antibodies of the
present invention can readily be incorporated into one of the
established kit formats which are well known in the art.
[0094] An antibody is said to be in "pharmaceutically or
pharmacologically acceptable form" if its administration can be
tolerated by a recipient patient. The antibodies of the present
invention can be formulated according to known methods of preparing
pharmaceutically useful compositions, whereby these materials, or
their functional derivatives, are combined with a pharmaceutically
acceptable carrier vehicle. Suitable vehicles and their
formulation, inclusive of other human proteins, e.g., human serum
albumin, are described, for example, in Remington's Pharmaceutical
Sciences, 1980).
[0095] In order to form a pharmaceutically acceptable composition
which is suitable for effective administration, such compositions
will contain an effective amount of an antibody of the present
invention together with a suitable amount of carrier. Such carriers
include, but are not limited to saline, buffered saline, dextrose,
water, glycerol, ethanol, and a combination thereof. The carrier
composition may be sterile. The formulation should suit the mode of
administration. In addition to carriers, the antibodies of the
present invention may be supplied in humanized form.
[0096] Humanized antibodies may be produced, for example by
replacing an immunogenic portion of an antibody with a
corresponding, but non-immunogenic portion (i.e., chimeric
antibodies) (Robinson et al., International Patent Publication
PCT/US86/02269; Akira et al., European Patent Application 184,187;
Taniguchi, European Patent Application 171,496; Morrison et al.,
European Patent Application 173,494; Neuberger et al., PCT
Application WO 86/01533; Cabilly et al., European Patent
Application 125,023; Better et al., Science 240:1041-1043 (1988);
Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu et
al., J. Immunol. 139:3521-3526 (1987); Sun et al., Proc. Natl.
Acad. Sci. USA 84:214-218 (1987); Nishimura et al., Canc. Res.
47:999-1005 (1987); Wood et al., Nature 314:446-449 (1985)); Shaw
et al., J. Natl. Cancer Inst. 80:1553-1559 (1988).
[0097] The compositions of the present invention can also include
minor amounts of wetting or emulsifying agents, or pH buffering
agents. The composition can be a liquid solution, suspension,
emulsion, tablet, pill, capsule, sustained release formulation or
powder. The composition can be formulated as a suppository with
traditional binders and carriers such as triglycerides. Oral
formulations can include standard carriers such as pharmaceutically
acceptable mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc.
[0098] In a preferred embodiment of the present invention, the
compositions are formulated in accordance with routine procedures
for intravenous administration to a subject. Typically, such
compositions are carried in a sterile isotonic aqueous buffer. As
needed, a composition may include a solubilizing agent and a local
anesthetic. Generally, the components are supplied separately or as
a mixture in unit dosage form, such as a dry lyophilized powder in
a sealed container with an indication of active agent. Where the
composition is administered by infusion, it may be provided with an
infusion container with a sterile pharmaceutically acceptable
carrier. When the composition is administered by injection, an
ampoule of sterile water or buffer may be included to be mixed
prior to injection.
[0099] The therapeutic compositions may also be formulated in salt
form. Pharmaceutically acceptable salts include those formed with
free amino groups, such as those derived from hydrochloric,
phosphoric, acetic, oxalic and tartaric acids, or formed with free
carboxyl groups such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides. isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0100] The dosage of the administered agent will vary depending
upon such factors as the patient's age, weight, height, sex,
general medical condition, previous medical history, etc. In
general, it is desirable to provide the recipient with a dosage of
the antibody which is in the range of from about 1 .mu.g/kg to 10
mg/kg (body weight of patient), although a lower or higher dosage
may be administered. Suitable ranges for intravenous administration
is typically about 20-500 .mu.g of active compound per kilogram
body weight. Effective doses may be extrapolated from dose-response
curves derived from in vitro and in vivo animal model test
systems.
[0101] Since highly purified proteins are now available, X-ray
crystallography and NMR-imaging techniques can be used to identify
the structure of the enzyme. Utilizing such information, computer
modeling systems are now available that allows one to "rationally
design" an agent capable of binding to a defined structure
(Hodgson, Biotechnology 8:1245-1247 (1990)), Hodgson, Biotechnology
9:609-613 (1991)). As used herein, an agent is said to be
"rationally designed" if it is selected based on a computer model
of nNOS-II.
[0102] In another embodiment of the present invention, methods are
provided for modulating the translation of RNA encoding nNOS-II
protein in the cell. Specifically, said method comprises
introducing into a cell a DNA sequence which is capable of
transcribing RNA which is complimentary to the RNA encoding the
nNOS-II protein. By introducing such a DNA sequence into a cell,
antisense RNA will be produced which will hybridize and block the
translation of the nNOS-II protein. Antisense cloning has been
described by Rosenberg et al., Nature 313:703-706 (1985), Preiss et
al., Nature 313:27-32 (1985), Melton, Proc. Natl. Acad. Sci. USA
82:144-148 (1985) and Kim et al., Cell 42:129-138 (1985).
[0103] Transcription of the introduced DNA will result in multiple
copies of antisense RNA which will be complimentary to the nNOS-II.
By controlling the level of transcription of antisense RNA, and the
tissue specificity of expression, one skilled in the art can
regulate the level of activity of nNOS-II in specific cells within
a patient.
[0104] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
previously described methods and assays.
[0105] All essential publications mentioned herein are hereby
incorporated by reference.
[0106] In order that those skilled in the art can more fully
understand this invention, the following examples are set forth.
These examples are included solely for the purpose of illustration,
and should not be considered as expressing limitations unless so
set forth in the appended claims.
EXAMPLES
[0107] In the following examples and protocols, restriction
enzymes, ligase, labels, and all commercially available reagents
were utilized in accordance with the manufacturer's
recommendations. The cell and molecular and protein purification
methods utilized in this application are established in the art and
will not be described in detail. However, standard methods and
techniques for isolation, purification, labeling, and the like, as
well as the preparation of standard reagents may be performed
essentially in accordance with Molecular Cloning: A Laboratory
Manual, 2nd ed., edited by Sambrook, Fritsch & Maniatis, Cold
Spring Harbor Laboratory, 1989, and the revised third edition
thereof, or as set forth in the literature references cited and
incorporated herein. Methodologic details may be readily derived
from the cited publications.
Example 1
Isolation, Purification and Characterization of nNOS-II
[0108] Using the following materials and methods, substantially
pure nNOS-II protein was prepared and purified from crude
supernatant preparations from rat cerebellum, and characterized as
containing calmodulin-independent NOS activity capable of
catalyzing the oxidation of an arginine-rich nonapeptide, BK, to
produce NO.
[0109] Materials: BK, [Thi.sup.5,8,D-Phe.sup.7]-BK, and
[N-adamantaneacetyl-D-Arg.sup.0, Hyp.sup.3, Thi.sup.5,8,
D-Phe.sup.7]-BK were purchased from American Peptide Co. (San
Diego, Calif.). Cit.sup.1-BK was synthesized (University of Iowa,
Protein Structure Facility) by solid phase synthesis using
2-chlorotrityl chloride resin (Barbos et al., Int. J. Peptide
Protein Res. 37:513-520 (1991)). The synthetic peptide Cit.sup.1-BK
was greater than 98% pure by HPLC, and gave m/z 1061.3 by laser
desorption mass spectrometry. (6R)-5,6,7,8-Tetrahydrobiopterin
(BH.sub.4) was from Biochemical Research Inc. (Natick, Mass.).
L-arginine, N.sup.G-methyl-L-arginine (L-NMA),
N.sup.G-nitro-L-arginine (L-NNA), 2',5'-ADP-agarose and other
reagents were purchased from Sigma Chemical Co. (St Louis,
Mo.).
[0110] Enzyme Purification: Ten cerebella taken from Sprague Dailey
rats (male, 250-350 g) were homogenized in 50 mL of ice-cold buffer
A {10 mM Tris-HCl (pH 7.5) containing 1 mM DTT, 1 mM EDTA}. All
subsequent purification procedures were carried out at 4.degree. C.
The homogenate was centrifuged at 100,000.times.g for 120 min, and
the supernatant was loaded onto a 25 mL DEAE-agarose column
equilibrated with buffer A. The column was washed with 50 mL buffer
A and eluted with a 200 mL linear gradient of 0-500 mM NaCl in
buffer A. Fractions (4 mL) were collected and assayed for enzyme
activity.
[0111] Elution profiles similar to those observed by Bredt et al.
(Proc. Natl Acad. Sci. USA 87:682-685 (1990)) reproducibly gave two
calmodulin-dependent NOS activity peaks with L-arginine as
substrate (see FIG. 1, fractions 8-21 (.largecircle.). However,
only one of these (FIG. 1, fractions 15-21 (.circle-solid.))
displayed calmodulin-independent NOS activity with BK as substrate.
Protein absorbance at 280 nm (FIG. 1, .quadrature.) from rat
cerebellum was used to monitor the eluting process.
[0112] Fractions representing the active peak for BK from the
DEAE-agarose column were pooled and concentrated to 15 mL by
membrane ultrafiltration, and loaded onto a 5 mL, 2',5'-ADP-agarose
column equilibrated with buffer B {10 mM Tris-HCl (pH 7.0)
containing 1 mM DTT, 1 mM EDNA, and 10% glycerol}. The column was
subsequently washed with 20 mL of buffer B, 10 mL of 0.5 M NaCl in
buffer B, 10 mL of 0.5 mM NADH in buffer B, 10 mL of 0.5 mM
NADP.sup.+ in buffer B, and 20 mL of buffer B. The NOS activity was
finally eluted with 10 mL of 10 mM NADPH in buffer B.
[0113] The affinity eluate was concentrated to 1.5 mL and loaded
onto a 5 mL DEAE-agarose column equilibrated with buffer B. The
column was washed with 20 mL buffer B and eluted with a 100 mL
linear gradient of 0-300 mM NaCl in buffer B. Fractions (2 mL) were
collected and assayed for enzyme activity, and active fractions (8
mL) were pooled and concentrated to 0.2-0.5 mL with an Amicon PM-30
membrane to afford a 6,360-fold purified enzyme preparation. The
thus-purified, novel constitutive neuronal NOS was designated as
nNOS-II to differentiate it from the NOS isoform (nNOS) previously
described by Bredt et al., supra (1990).
[0114] The purification of nNOS-II is summarized in Table 2:
2TABLE 2 Purification of nNOS-II from rat cerebellum.* Specific
Activity Total Protein Total Activity (nmol/min/mg) Purification
Step (.mu.g) (nmol/min) protein) Recovery (%) Factor (fold) Cytosol
356,640 44.18 0.19 100 1 DEAE- 69,200 27.20 0.39 61.6 2 agarose
ADP-agarose 66 19.10 289.1 43.2 1,522 DEAE- 4 4.64 1,208.3 10.5
6,360 agarose *Values of final specific activities, recovery and
fold-purification represent the means of three enzyme preparations.
Enzyme activity was measured spectophotometically. BK (100 .mu.M)
was used for enzyme assay during enzyme purification, and enzymatic
reactions were initiated by the addition of protein. Protein was
determined using the Bradford protein assay.
[0115] NOS Activity Assay: NOS activity was determined
spectrophotometrically by the rapid and quantitative oxidation of
oxyhemoglobin to methemoglobin (Feelish et al., Eur. J. Pharmacol.
139:19-30 (1987); Olken et al., Biochem. Biophys. Res. Commun.
177:828-833 (1991)).
[0116] Like previously characterized nNOS from cerebellum, nNOS-II
requires NADPH, FAD, FMN, Ca.sup.2+, and BH.sub.4 with BK as
substrate. Thus, reaction mixtures for NOS assays containing 50 mM
Tris-HCl buffer (pH 7.5), were optimized to contain 4 .mu.M
oxyhemoglobin, 100 .mu.M NADPH, 1 mM CaCl.sub.2, 10 .mu.M FAD, 10
.mu.M FMN, 20 .mu.M BH.sub.4, 150 .mu.M DTT, 100 .mu.M BK or
L-arginine and 0.1-3 .mu.g enzyme in a final volume of 0.5 mL.
[0117] nNOS-II has stabilities similar to nNOS. In addition,
nNOS-II also oxidizes L-arginine, but only when calmodulin is added
along with the other cofactors. Thus, calmodulin (10 .mu.g/mL) was
added to the reaction mixtures when L-arginine was used as
substrate.
[0118] Determinations of Molecular Weight: The molecular mass of
the denatured purified enzyme was determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be
approximately 105 kD (see, FIG. 2). The apparent native molecular
mass of the enzyme was approximately 230 kD by gel filtration,
indicating that nNOS-II is a homodimeric protein.
[0119] The native molecular weight of the purified enzyme was
estimated by analytical gel filtration chromatography carried out
using an Alltech Macrosphere 150 column (7 .mu.m, 4.6.times.25 cm)
and a mobile phase of 10 mM Tris-HCl buffer (pH 7.5) containing 1
mM DTT and 0.2 M NaCl, which was used to equilibrate the column and
to elute protein samples at a flow rate of 0.5 mL/min. Eluting
protein peaks (retention volumes, R.sub.v) were monitored at 280
nm. Standard proteins (Mr) used (as shown in FIG. 2) were bovine
thyroglobulin (669,000, R.sub.v 1.83 mL), horse spleen apoferritin
(443,000, R.sub.v 1.97 mL), sweet potato .beta.-amylase (200,000,
R.sub.v 2.28 mL), yeast alcohol dehydrogenase (150,000, R.sub.v
2.47 mL), and bovine serum albumin (66,000, R.sub.v 2.78 mL).
[0120] The peak (R.sub.v 2.25 mL) corresponding to the eluted
enzyme (230,000) was collected and assayed for NOS activity.
[0121] Kinetic Determinations: Since BK contains both N- and
C-terminal arginines, it was possible that BK could give rise to NO
plus three different nonapeptide products including: Cit.sup.1-BK,
Cit.sup.9-BK and Cit.sup.1,Cit.sup.9-BK. HPLC and amino acid
sequencing were used to isolate and identify the nonapeptide
product formed when nNOS-II oxidized BK. Kinetic experiments were
conducted using the standard enzyme assay described above.
[0122] a) Isolation of Enzymatic Products by High Performance
Liquid
[0123] Chromatography (HPLC): Enzymatic reaction mixtures contained
0.5 .mu.g purified enzyme, 50 mM Tris-HCl buffer (pH 7.5), 100
.mu.M NADPH, 20 .mu.M FAD, 20 .mu.M FMN, 1 mM CaCl.sub.2, 50 .mu.M
tetrahydrobiopterin, and 100 .mu.M substrate in final volumes of
0.5 mL. Reaction mixtures were incubated for 120 min at 37.degree.
C., and transferred to microconcentrators (Mr cut-off 10,000,
Bio-Rad) and centrifuged to remove the enzyme. Fractions through
the concentrator membrane were subjected to HPLC analyses. HPLC was
performed with a Shimadzu LC-10AD pump and a SPD-M6A photodiode
array UV-Vis detector set at 214-219 nm. Samples of 15 .mu.L were
resolved on a .mu.Bondapak C18 column (Waters; 10 mm; 3.9.times.300
mm, inside diameter) preceded by a guard column (3.9.times.20 mm)
at a flow rate of 1 mL/min. The mobile phase consisted of mixtures
of: A. 0.1% trifluoroacetic acid in water; B. 0.095%
trifluoroacetic acid in acetonitrile.
[0124] Elution was achieved with the following gradients: 0-15 min,
0-15% linear gradient of B; 15-35 min, 15-20% linear gradient of B;
and 35-40 min, 20-0% linear gradient of B. Retention times for BK,
Cit.sup.1-BK, and Cit.sup.1,Cit.sup.9-BK are 22.8 min, 24.3 min,
and 26.3 min, respectively.
[0125] For amino acid sequencing, reaction mixture samples of 200
.mu.L were injected, and peaks at 26.3 min were collected and
concentrated. Incubations without substrate and without purified
enzyme were used and analyzed as controls.
[0126] b) Amino Acid Sequencing:
[0127] Amino acid sequencing was determined by automated
microsequencing with Edman degradation reactions on a 475A
Sequencer (Applied Biosystems, Inc.) in the University of Iowa,
Protein Structure Facility. The enzymatic product (2 .mu.g) from
HPLC (see, FIGS. 3A and B) was sequenced in duplicate to confirm
the peptide sequence.
[0128] c) Kinetic Data:
[0129] To determine K.sub.M values for BK, Cit.sup.1-BK and
L-arginine, the substrate concentrations used were 3.125, 6.25,
12.5, 25, and 50 .mu.M. L-NMA and L-NNA, at concentrations of 0,
3.125, 6.25, 12.5, 25, and 50 .mu.M, were used to measure Ki values
in the presence of above concentrations of BK.
N-adamantaneacetyl-[D-Arg.sup.0, Hyp.sup.3, Thi.sup.5,8,
D-Phe.sup.7]-BK, at concentrations of 0, 0.625, 1.25, 2.5, 5, and
10 .mu.M, was used to determine its Ki value in the presence of the
concentrations of BK given above. Kinetic data were calculated by
fitting experimental data to the EZ-FIT program (Perrella, Anal
Biochem. 174:437-447 (1988)).
[0130] The only peptide product obtained during the nNOS-II
oxidation of BK was Cit.sup.1,Cit.sup.9-BK, where both N- and
C-terminal BK-arginines were converted to their corresponding
citrullines (FIG. 3A). The synthetic nonapeptide Cit.sup.1-BK in
which N-terminal arginine was replaced with citrulline was also a
substrate for nNOS-II, and it, too, gave Cit.sup.1,Cit.sup.9-BK
(FIG. 3B). Difficulties encountered in synthesizing Cit.sup.9-BK
precluded its use in the present investigations.
[0131] Apparent K.sub.M values for BK and Cit.sup.1-BK were 8.5 and
6.2 .mu.M, respectively, while apparent V.sub.max values for BK and
Cit.sup.1-BK were 1.2 and 1.6 .mu.mol/min/mg protein, respectively.
The kinetic results suggest that neither Cit.sup.1-BK nor
Cit.sup.9-BK are likely to accumulate as products during the course
of nNOS-II oxidations of BK.
[0132] By comparison, the apparent K.sub.M of nNOS-II for
L-arginine was 10.6 .mu.M, slightly higher than the reported value
of 2 .mu.M for previously described nNOS (Bredt et al., Proc. Natl
Acad. Sci. USA 87:682-685 (1990)). With L-arginine as substrate,
nNOS-II and nNOS display apparent V.sub.max values of 0.85 and 0.96
.mu.mol/min/mg protein, respectively.
[0133] With nNOS-II and BK as substrate, typical NOS inhibitors,
L-NMA and L-NNA, are reversible, competitive inhibitors with
apparent Ki values of 8.6 .mu.M and 23.8 .mu.M, respectively. These
Ki values are, however, significantly different from previously
established Ki values for L-NMA of 1.4 .mu.M and L-NNA of 4.4 .mu.M
with L-arginine as substrate for nNOS (Nathan, FASEB J. 6:3051-3064
(1992); Marletta, J. Biol. Chem. 268:12231-12234 (1993); Knowles et
al., Biochem. J. 298:249-258 (1994); Griffith et al., Annu. Rev.
Physiol. 57:707-736 (1995)).
[0134] In addition, known potent specific B2 receptor antagonists
[N-adamantaneacetyl-D-Arg.sup.0, Hyp.sup.3, Thi.sup.5,8,
D-Phe.sup.7]-BK and [Thi.sup.5,8, D-Phe.sup.7]-BK (Lammek et al.,
Peptides 11:1041-1043 (1990); Lammek et al., J. Phar. Pharmacol.
43:887-888 (1988); Austin et al., J. Physiol. 478:351-356 (1994))
were examined for their possible effects with regard to nNOS-II
activity. While [N-adamantaneacetyl-D-Arg.- sup.0, Hyp.sup.3,
Thi.sup.5,8, D-Phe.sup.7]-BK competitively inhibited nNOS-II
activity on BK, with an apparent Ki of 2.5 mM, [Thi.sup.5,8,
D-Phe.sup.7]-BK had no effect on nNOS-II activity over a
concentration range of 0.5-100 mM.
[0135] In view of these findings, it is believed that natural
endogenous peptide substrates other than BK exist for nNOS-II. It
is also believed that oligopeptide-utilizing endothelial and
macrophage NOSs will be found. The broad implications of this
discovery are that arginine-rich peptides of greater or lesser size
than BK can serve as NOS substrates to form NO, and that natural or
synthetic peptides, oligopeptides or proteins can function as
NOS.
[0136] Although the present invention has been described with
reference to the presently preferred embodiments, the skilled
artisan will appreciate that various modifications, substitutions,
omissions and changes may be made without departing from the spirit
of the invention. Accordingly, it is intended that the scope of the
present invention be limited only by the scope of the following
claims, including equivalents thereof.
Example 2
Comparison of Reaction Rates with Bradykinin Analogs
[0137] Relative reaction rates were compared with a series of BK
analogs. As expected, most of the BK analogs studied served as
substrates for nNOS-II. Table 11 shows the relative activities of
the arginine-containing peptides as substrates for nNOS-II.
Different than that for NOS.sub.NOC, poly-arginine only showed 30%
activity for nNOS-II comparing to that of arginine. However,
nonapeptide BK showed more activity than arginine; Moreover, the
octapeptide, des-Arg.sup.1-BK, in which the N-terminal arginine is
removed had activity similar to that of arginine. Another
octapeptide, des-Arg.sup.9-BK, in which the C-terminal arginine is
removed had activity about one-fold higher than that of arginine.
The heptapeptide BK fragment 1-7 decreased its activity by 30%
while BK fragment 1-5 had about 50% activity of that for BK. When
the N-terminal arginine was replaced by lysine, nonapeptide
[Lys.sup.1]-BK had similar activity to that for BK, while BK with
an additional N-terminal lysine, decapeptide Lys-BK, had almost the
same activity. However, Lys-BK with an additional methonine, the
undecapeptide Met-Lys-BK, showed no activity at all. Although the
length of peptides, the position of arginine in the peptides, and
the presence of certain amino acids such as lysine may affect the
activities for nNOS-II as substrates, there is no clear
relationship between the peptide length or the position of arginine
and NOS activity. These preliminary results indicate that BK and
analogs can directly serve as substrates for nNOS-II, although
structure-activity relationship could not finally established based
on this study.
3TABLE 3 Arginine-Containing Peptides as Substrates for
nNOS-II.sup.a Relative Peptide Amino acid sequence activity (%)
L-Arginine N/A.sup.b 100.sup.c Poly-arginine N/A 30 (M.sub.r 5,000)
BK Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 125 Des-Arg1-BK
Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 94 Des-Arg9-BK
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe 180 BK fragment 1-7
Arg-Pro-Pro-Gly-Phe-Ser-Pro 80 BK fragment 1-5 Arg-Pro-Pro-Gly-Phe
61 BK fragment 2-7 Pro-Pro-Gly-Phe-Ser-Pro 0 [Lys1]-BK
Lys-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 113 Lys-BK
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro- 116 Phe-Arg Ile-Ser-BK
Ile-Ser-Arg-Pro-Pro-Gly-Phe-Ser-Pro- 110 Phe-Arg Met-Lys-BK
Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro- 0 Phe-Arg .sup.aStandard
enzyme assay was carried out to measure initial velocity of the
enzymatic reaction. Purified nNOS-II 0.2 .mu.g (specific activity
fof BK, 280 nmole NO formed/min/mg protein) was used for the
assays. Substrate concentrations were 100 .mu.M except for
poly-arginine used with 5 .mu.g/ml. .sup.bNot applicable.
.sup.cActivity for arginine is expressed as 100%. The values are
the means of three measurements.
Example 3
[0138] The following is hereby expressly incorporated in its
entirety by reference:
[0139] Chen, Yijun and Rosazza, John P. N., "Oligopeptides as
Substrates and Inhibitors for a New Constitutive Nitric Oxide
Synthase from Rat Cerebellum", Biochemical and Biophysical Research
Communications, 224:303-308 (1996), Article No. 1025.
Oligopeptides as Substrates and Inhibitors for a New Constitutive
Nitric Oxide Synthase from Rat Cerebellum
Materials and Methods
[0140] Materials, BK [Thi.sup.5,8, D-Phe.sup.7]-BK, and
[N-adamantaneacetyl-D-Arg.sup.0, Hyp.sup.3, Thi.sup.5,8,
D-Phe.sup.7]-BK were purchased from American Peptide Co. (San
Diego, Calif.). Cit.sup.1-BK was synthesized (University of Iowa,
Protein Structure Facility) by solid phase synthesis using
2-chlorotrityl chloride resin (9). The synthetic peptide
Cit.sup.1-BK was greater than 98% pure by HPLC, and gave m/z 1061.3
by laser desorption mass spectrometry.
(6R)-5,6,7,8-Tetrahydrobiopterin (BH.sub.4) was from Biochemical
Research Inc. (Natick, Mass.), L-Arginine,
N.sup.G-methyl-L-arginine (L-NMA), N.sup.G-nitro-L-arginine
(L-NNA), 2',5'-ADP-agarose and other reagents were purchased from
Sigma Chemical Co. (St. Louis, Mo.).
[0141] NOS activity assay. NOS activity was determined
spectrophotometrically by the rapid and quantitative oxidation of
oxyhemoglobin to methemoglobin (10,11). Optimized reaction mixtures
for NOS assays contained 50 mM Tris-HCl buffer (pH 7.5), and were
optimized to contain 4 .mu.M oxyhemoglobin. 100 .mu.M NADPH. 1 mM
CaCl.sub.2, 10 .mu.M FAD. 10 .mu.M FMN, 20 .mu.M BH. 150 .mu.M DTT,
100 .mu.M BK or L-arginine and 0.1-3 .mu.g enzyme in a final volume
of 0.5 ml. Calmodulin (10 .mu.g/ml) was added to the reaction
mixtures when L-arginine was used as substrate.
[0142] Enzyme purification. Ten cerebella taken from Sprague Dawley
rats (male. 250-350 g) were homogenized in 50 ml of ice-cold buffer
A [10 mM Tris-HCl (pH 7.5) containing 1 nM DTT, 1 mM EDTA), and all
subsequent purification procedures were carried out at 4.degree. C.
The homogenate was centrifuged at 100,000.times.g for 120 min. and
the supernatant was loaded onto a 25 ml DEAE-agarose column
equilibrated with buffer A. The column was washed with 50 ml buffer
A and eluted with a 200 ml linear gradient of 0-500 mM NaCl in
buffer A. Fractions (4 ml) were collected and assayed for enzyme
activity. Fractions representing the active peak for BK from the
DEAE-agarose column were pooled and concentrated to 15 ml by
membrane ultrafiltration, and loaded onto a 5 ml, 2',5'-ADP-agarose
column equilibrated with buffer B [10 mM Tris-HCl (pH 7.0)
containing 1 mM DTT, 1 mM EDTA, and 10% glycerol]. The column was
subsequently washed with 20 ml of buffer B, 10 ml of 0.5 mM NADH in
buffer B, 10 ml of 0.5 mM NADP.sup.- in buffer B, and 20 ml of
buffer B. The NOS activity was finally eluted with 10 ml of 10 mM
NADPH in buffer B. The affinity eluate was concentrated to 1.5 ml
and loaded onto a 5 ml DEAE-agarose column equilibrated with buffer
B. The column was washed with 20 ml buffer B and eluted with a 100
ml linear gradient of 0-300 mM NaCl in buffer B. Fractions (2 ml)
were collected and assayed for enzyme activity, and active
fractions (8 ml) were pooled and concentrated to 0.2-0.5 ml with an
Amicon PM-30 membrane.
[0143] Determinations of molecular weight. The molecular weight of
denatured purified enzyme was determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native
molecular weight of the purified enzyme was estimated by analytical
gel filtration chromatography carried out using an Alltech
Macrosphere 150 column (7 .mu.m, 4.6.times.25 cm) and a mobile
phase of 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM DTT and 0.2
M NaCl which was used to equilibrate the column and to elute
protein samples at a flow rate of 0.5 ml/min. Eluting protein peaks
(retention volumes, R.sub.v) were monitored at 280 nm. Standard
proteins (M.sub.r) used were bovine thyroglobulin (669,000, R.sub.v
1.83 ml), horse spleen apoferritin (443,000, R, 1.97 ml), sweet
potato .beta.-amylase (200,000, R.sub.v 2.28 ml), yeast alcohol
dehydrogenase (150,000, R.sub.v 2.47 ml), and bovine serum albumin
(66,000, R.sub.v 2.78 ml). The peak (R.sub.v 2.25 ml) corresponding
to the eluted enzyme (230,000) was collected and assayed for NOS
activity.
[0144] Kinetic determinations. Kinetic experiments were conducted
using the standard enzyme assay described above. To determine Km
values for BK, Cit.sup.1-BK and L-arginine, substrate
concentrations used were 3.125, 6.25, 12.5, 25 and 50 .mu.M. L-NMA
and L-NNA with concentrations of BK.
N-adamantaneacetyl-[D-Arg.sup.0, Hyp.sup.3, Thi.sup.5,8,
D-Phe.sup.7]-BK with concentrations of 0, 0.625, 1.25, 2.5, 5 and
10 .mu.M was used to determine its Ki value in the presence of the
concentrations of BK given above. Kinetic data were calculated by
fitting experimental data to EZ-FIT program (12).
[0145] Isolation of enzymatic products by high performance liquid
chromatography (HPLC). Enzymatic reaction mixtures contained 0.5
.mu.g purified enzyme, 50 mM Tris-HCl buffer (pH 7.5), 100 .mu.M
NADPH, 20 .mu.M FAD, 20 .mu.M FMN. 1 mM CaCl.sub.2, 50 .mu.M
tetrahydrobiopterin, and 100 .mu.M substrate in final volumes of
0.5 ml. Reaction mixtures were
4TABLE 1 Purification of nNOS-II from Rat Cerebellum* Total Total
Purification protein activity Specific activity Recovery factor
Step (.mu.g) (nmol/min) (nmol/min/mg protein) (%) (fold) Cytosol
356,640 44.18 0.19 110 1 DEAE-agarose 69,200 27.20 0.39 61.6 2
ADP-agarose 66 19.10 289.1 43.2 1,522 DEAE-agarose 4 4.64 1,208.3
10.5 6,360 *Values of final specific activities, recovery, and
fold-purification were the means of three enzyme preparations.
Enzyme activity was measured spectrophotometrically. BK (100 .mu.M)
was used for enzyme assay during enzyme purification, and enzymatic
reactions were initiated by the addition of protein. Protein was
determined with the Bradford #protein Assay.
[0146] incubated for 120 min at 37.degree. C., and transferred to
microconcentrators (Mr cut-off 10,000, Bio-Rad) and centrifuged to
remove the enzyme. Fractions through the concentrator membrane were
subjected to HPLC analyses. HPLC was performed with a Shimadzu
LC-10AD pump and a SPD-M6A photodiode array UV-Vis detector set at
214-219 nm. Samples of 15 .mu.l were resolved on a .mu.Bondapak
C18-column (Waters: 10 .mu.m; 3.9.times.300 mm, inside diameter)
preceded by a guard column (3.9.times.20 mm) at a flow rate of 1
ml/min. The mobile phase consisted of mixtures of: A. 0.1%
trifluoroacetic acid in water; B. 0.095% trifluoroacetic acid in
acetonitrile. Elution was achieved with the following gradients:
0-15 min, 0-15% linear gradient of B; 15-35 min, 15-20% linear
gradient of B; and 35-40 min. 20-0% linear gradient of B. Retention
times for BK, Cit.sup.1-BK, and Cit.sup.1.Cit.sup.9-BKare 22.8 min,
24.3 min, and 26.3 min. respectively. For amino acid sequencing,
reaction mixture samples of 200 .mu.l were injected and peas at
26.3 min were collected and concentrated. Incubations without
substrate and without purified enzyme were used and analyzed as
controls.
[0147] Amino acid sequencing. Amino acid sequencing was determined
by automated microsequencing with Edman degradation reactions on a
475A Sequencer (Applied Biosystems, Inc.) in the University of
Iowa. Protein Structure Facility. The enzymatic product (2 .mu.g)
from HPLC (FIG. 3) was sequenced in duplicate to confirm the
peptide sequence.
Results and Discussion
[0148] Crude supernatant preparations from rat cerebellum contained
calmodulin-independent, NOS activity capable of catalyzing the
oxidation of BK to produce NO. Since the first step in purifying
arginine-utilizing neuronal nitric oxide synthase (nNOS) from rat
cerebellum (13) used weak anion exchange chromatography
(DEAE-agarose), this approach was taken to partially purify the
BK-utilizing NOS enzyme, Elution profiles similar to those observed
by Bredt and Snyder (13) reproducibly gave two calmodulin-dependent
NOS activity peaks with L-arginine as substrate. Only one of these
(FIG. 1, fractions 1.5-21) displayed calmodulin-independent NOS
activity with BK as substrate. The calmodulin-independent and BK
active peak was further subjected to 2',5'-ADP-agarose affinity
chromatography and a second DEAE-agarose chromatographic step to
afford a 6,360-fold purified enzyme preparation (Table 1). The new
constitutive neuronal NOS, designated as nNOS-II to differentiate
it from previously describe nNOS (13), has, a molecular mass of 105
KD by SDS-PAGE (FIG. 2). The apparent native molecular mass of the
enzyme was 230 kD by gel filtration, indicating that nNOS-II is a
homodimeric protein. Like previously characterized nNOS from
cerebellum, nNOS-II requires NADPH, FAD, FMN Ca.sup.2+, and
BH.sub.4 with BK as substrate. nNOS-II has stabilities similar to
nNOS. nNOS-II also oxidizes L-arginine, but only when calmodulin is
added along with the other cofactors.
[0149] Since BK contains both N- and C-terminal arginines, it was
possible that BK could give rise to NO plus three different
nonapeptide products including: Cit.sup.1-BK, Cit.sup.9-BK and
Cit.sup.1,Cit.sup.9-BK. HPLC and amino acid sequencing were used to
isolate and identify the nonapeptide product formed when nNOS-II
oxidized BK. The only peptide product obtained during the nNOS-II
oxidation of BK was Cit.sup.1,Cit.sup.9-BK, where both N- and
C-terminal BK-arginines were converted to their corresponding
citrullines (FIG. 3A). The synthetic nonapeptide Cit.sup.1-BK in
which N-terminal arginine was replaced with citrulline was also a
substrate for nNOS-II, and it too gave Cit.sup.1,Cit.sup.9-BK (FIG.
3B). Difficulties encountered in synthesizing Cit.sup.9-BK
precluded its use in ,these studies. Apparent K.sub.M values for BK
and Cit.sup.1-BK were 8.5 and 6.2 .mu.M. respectively while
apparent V.sub.max values for BK and Cit.sup.1-BK were 1.2 and 1.6
.mu.mol/min/mg protein. respectively. The kinetic results Suggest
that neither Cit.sup.1-BK nor Cit.sup.9-BK are likely to accumulate
as products during the course of nNOS-II oxidations of BK. The
apparent Km of nNOS-II for L-arginine was 10.6 .mu.M, slightly
higher than the reported value of 2 .mu.M for previously described
nNOS (13). With L-arginine as substrate nNOS-II and nNOS display
apparent V.sub.max values of 0.85 and 0.96 .mu.mol/min/mg protein,
respectively.
[0150] With nNOS-II and BK as substrate, typical NOS inhibitors
(1-4). L-NMA and L-NNA, were reversible, competitive inhibitors
with apparent Ki values of 8.6 .mu.M and 23.8 .mu.M respectively.
These Ki values are significantly different than previously
established Ki values for L-NMA of 1.4 .mu.M and L-NNA of 4.4 .mu.M
with L-arginine as substrate for nNOS (1-4). Known potent specific
B12receptor antagonists [N-adamantaneacetyl-D-Aro.sup.0, Hyp.sup.3,
Thi.sup.5,8, D-Phe.sup.7]-BK and (Thi.sup.5,8. D-Phe.sup.7]-BK
(14-16) were examined for their possible effects vs. nNOS-II
activity. While [N-adamantaneacetyl-D-Arg.su- p.0, Hyp.sup.3,
Thi.sup.5,8, D-Phe.sup.7]-BK competitively inhibited nNOS-II
activity vs. BK with an apparent Ki of 2.5 .mu.M, [Thi.sup.5,8,
D-Phe.sup.7]-BK had no effect nNOS-II activity over a concentration
range of 0.5-100 .mu.M.
[0151] This study reports the discovery of a new constitutive
neuronal NOS (nNOS-II) in rat cerebellum that directly oxidizes
both L-arginine (calmodulin dependent) and oligopeptides
(calmodulin independent) as sources of NO (FIG. 4). Peptide
products, and some catalytic and kinetic properties mere defined
for nNOS-II. Although NO is apparently formed from dipeptides like
Arg-Arg and Arg-Phe by crude enzyme preparations from cultured
endothelial and macro-phage cells (17-19), this is the first report
describing the oxidation of oligopeptides by a mammalian NOS, and
the inhibition of an NOS by peptide antagonists. Our results
suggest that natural endogenous peptide substrates other than BK
may exist for nNOS-II, and that oligopeptide utilizing endothelial
and macrophage NOSs remain to be identified. The broad implications
of this work are that arginine-rich peptides of greater or lesser
size than BK can serve as NOS substrates to form NO, and that
natural or synthetic peptide antagonists can function as NOS
inhibitors.
ACKNOWLEDGMENTS
[0152] The authors thank Professors Robert J. Linhardt and Michael
W. Duffel for their valuable comments and, and YC thanks the Center
for Biocatalysis and Bioprocessing for fellowship support.
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