US 5,268,465 AGrant

Purification and Molecular Cloning of Nitric Oxide Synthase

Issue Date:1993-12-07

•11 Claims

•3 Drawing Sheets

Abstract

A method of purifying calmodulin-dependent nitric oxide synthase provides a homogeneous preparation of the enzyme. The enzyme is used to raise antibodies which are a useful immunohistochemical reagent. The antibodies localize calmodulin-dependent nitric oxide synthase to a number of anatomical sites, including retina, intestine, adrenal gland, and vasculature. However, activated macrophages, which are known to possess a nitric oxide producing activity, do not display an immunoreactive protein of appropriate size on Western blots using the antibodies. Nucleotide sequences encoding calmodulin-dependent nitric oxide synthase indicate a novel sequence with a flavin binding site consensus sequence.

Metadata

Assignee

The Johns Hopkins University

Inventors

David S. Bredt
Solomon H. Snyder

Application Information

Application Number:US 6420028

Filing Date:1991-01-18

Priority Date:1991-01-18

Art Unit:184

Classifications

IPC:

C12N 1500

Field of Search:

5364352769.1

Patent Drawings (3 sheets)

Description

Background of the Invention

Endothelium-derived relaxing factor, a labile substance formed by endothelial cells, which mediates vasodilation, has been shown to be identical to nitric oxide (NO) (Moncada et al., (1988) Biochem. Pharmacol. 37, pp. 2495-2501; Furchgott et al., (1988) FASEB J. 3, pp. 2007-2018; and Ignarro, L. J. (1989) FASEB J. 3, pp. 31-36). In addition to relaxing blood vessels, NO has multiple messenger functions as has been demonstrated in macrophages (Marletta et al., (1988) Biochemistry 27, pp. 8706-8711) and in brain tissue (Garthwaite et al., (1988) Nature 336, pp. 385-388; Knowles et al., (1989) Proc. Natl. Acad. Sci. USA, 86, pp. 5159-5162; and Bredt et al., (1989) Proc. Natl. Acad. Sci. USA, 86, pp. 9030-9033). NO appears responsible for the cytotoxic effects of macrophages and neutrophils (Hibbs et al., (1987) J. Immunol., 138, pp. 550-565).

Evidence that NO mediates functions of tissues as diverse as the brain, endothelium, and blood cells suggests a wide-spread role for NO as a messenger molecule. Localizing NO formation at a cellular level throughout the body would be greatly facilitated by immunohistochemical identification of NO synthase (NOS), the NO-forming enzyme. The use of NOS to supply deficient individuals with NO-forming ability would be expedited by the purification of the enzyme. In addition, the testing of populations for a genetic abnormality leading to deficient NO formation, such as in patients with migraines, hypertension, and coronary artery disease would be hastened by the isolation of the gene encoding NOS. Thus there is a need in the art for the biological tools for the characterization and manipulation of the NO-forming enzymatic apparatus.

Summary of the Invention

It is an object of the invention to provide a method of purifying calmodulin-dependent NOS.

It is another object of the invention to provide a purified preparation of calmodulin-dependent NOS.

It is still another object of the invention to provide a preparation of antibodies which is immunoreactive with calmodulin-dependent NOS.

It is yet another object of the invention to provide a cDNA molecule which encodes all or a portion of calmodulin-dependent NOS.

These and other objects of the invention are provided by one or more of the embodiments which are described below. In one embodiment of the invention a method of purifying calmodulin-dependent NOS is provided, which comprises the steps of:

contacting a preparation comprising calmodulin-dependent NOS with a solid matrix comprising NADPH or an NADPH analog; and

eluting calmodulin-dependent NOS from said solid matrix with NADPH or a soluble NADPH analog.

In another embodiment of the invention a purified preparation of calmodulin-dependent NOS is provided which has a specific activity of greater than about 500 nmoles citrulline/mg/min.

In yet another embodiment of the invention a preparation of antibodies is provided which is immunoreactive with brain-derived calmodulin-dependent NOS of a mammal but not with other proteins of said mammal, as determined by Western blotting.

In still another embodiment of the invention a cDNA molecule is provided which encodes all or a portion of calmodulin-dependent NOS, said molecule comprising between about 12 nucleotides and about 4,000 nucleotides.

The present invention provides the art with means to screen populations for genetic or acquired deficiencies in calmodulin-dependent NOS. In addition it provides the art with genes and proteins which can be used therapeutically to ameliorate the effects of genetic or acquired deficiencies in calmodulin-dependent NOS. The invention also provides a means of vasodilatation of blood vessels, for example those which may have become narrowed due to atherosclerosis.

Brief Description of the Drawings

FIG. 1 shows a representative purification of NO synthase on DEAE-cellulose.

FIG. 2A shows the dependence of NO Synthase activity on calcium, as a function of calmodulin concentration.

FIG. 2B shows the dependence of NO Synthase activity on NADPH as a function of calcium concentration.

FIG. 3 shows an SDS/PAGE analysis of purified NO synthase. A 7.5% polyacrylamide gel was stained with Coomassie blue. Lanes: A, molecular mass markers (Myosin, 200 kDa); phosphorylase b, 97 kDA; bovine serum albumin, 68 kDA; ovalbumin, 43 kDA; carbonic anhydrase, 29 kDA; B, 30 .mu.g of purified NO synthase. Silver staining displayed no additional protein bands with purified NO synthase.

Detailed Description

Nitric oxide synthase (EC #1.14.23) from brain has been purified to homogeneity, and its properties determined. It has been used to generate antibodies which have been used to localize the enzyme immunohistochemically. In addition, a cDNA encoding the NOS enzyme has been isolated and its sequence determined.

It is a finding of the present invention that NOS requires calmodulin for its enzyme activity. This contrasts with reported requirements of NOS isolated from activated macrophages, which require biopterins as a cofactor (Tayeh and Marletta; Kwon et al.). Macrophage-derived nitric oxide synthase has also been found to differ from that of brain-derived NOS immunochemically. Antisera raised against brain-derived NOS do not detect a similar sized protein in extracts of activated macrophages. The same antisera do, however, detect NOS in autonomic nerve fibers in the retina, in cell bodies and nerve fibers in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells. Thus it appears that there are at least two types of NOS proteins in mammalian tissues, one being calmodulin dependent and the other biopterin dependent.

According to the present invention, one can purify calmodulin-dependent NOS using column chromatography. Specifically, it has been found that greater than one-thousand-fold purification can be achieved using an affinity chromatography column. 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 binds 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) cellulose. Other ion exchange columns known in the art can also be used. The NOS of the present invention binds to DEAE-cellulose and can be eluted with a sodium chloride gradient. The greatest peak of activity elutes with between about 70 mM and about 145 mM sodium chloride. Combination of these two column chromatography steps on a cleared brain homogenate results in a homogeneous preparation, as determined both by silver staining of an SDS/PAGE-separated sample, as well as by Western blotting.

Tissues which can be used as a source of calmodulin-dependent NOS include brain, endothelial cells of blood vessels, and adrenal glands. In addition, recombinant host cells containing cDNA clones of the NOS gene can be used as a source of NOS for purification according to the present invention. Collection and processing of tissues can be done as is known in the art. Typically tissues will be homogenized in buffers containing protease inhibitors. Debris can be removed from the homogenate by centrifugation at about 20,000.times.g for 15 minutes. Stability of the enzyme is enhanced by storage in bovine serum albumin (1 mg/ml)/20% (vol/vol) glycerol at -70.degree. C. Stability is also enhanced in the presence of calmodulin.

Preparations of calmodulin-dependent NOS can be obtained which are homogeneous, according to techniques described above. Thus preparations having specific activities between about 500 and about 1000 nmol/mg/min are obtained. Preparations obtained according to the purification methods of the present invention are substantially free of contaminating proteins. Thus they are typically greater than 95% free of proteins of the same species source as the tissue from which they are extracted. Preferably, they are greater than 98% free of other proteins of the same species source. Using recombinant host cells to produce preparations of calmodulin-dependent NOS will also readily produce preparations which are substantially free of proteins of the same species source. Proteins which may be added back to a preparation to promote stability, such as calmodulin or bovine serum albumin, are not considered in the determination of purity. The presence of contaminating proteins can be determined using silver staining of polyacrylamide gels or Western blotting.

Antibody preparations are made according to techniques which are well known in the art. Both polyclonal and monoclonal antibodies are contemplated, production of both of which are well known. According to one method for obtaining a polyclonal antibody preparation, rabbits are immunized with a purified preparation of calmodulin-dependent NOS, as described above. The antiserum will preferably be affinity-purified by incubation with purified NOS and elution with 4M MgCl in 200 mM Tris-HCl buffer (pH 7.4). The eluate will desirably be dialysed against phosphate buffered saline with 0.1% Triton X100.

Antibodies can be used for immunohistochemical localization of NOS, or for quantitative assays on biological fluids or samples, such as in an enzyme-linked immunoadsorbent assay or radioimmunoassay. Such assays can determine if a tissue is producing an abnormally high or low amount of NOS.

cDNA molecules encoding calmodulin-dependent NOS are provided. The coding sequence of the gene is shown in SEQ ID NO:1. Whereas a particular nucleotide sequence is disclosed herein, other NOS-coding sequences are also encompassed by the invention, such as those which hybridize to the disclosed sequence, due to a difference in species source, allelic variations, or mutations introduced in the course of genetic manipulations. Thus other cDNA molecules which code for a closely related NOS are also contemplated. In addition, the cDNA molecule need not be complete in order to be useful. A portion of the cDNA can be used as a hybridization probe in order to quantitate mRNA expression, for example. Nucleotide probes are typically labeled with a detectable moiety such as a radioactive atom, or an enzyme. Whereas the entire gene-coding sequence is about 4 kb, sequences above about 12 to 15 nucleotides can be useful as hybridization probes. The cDNA sequence can also be used to hybridize to or amplify non-coding sequences. Thus the cDNA sequence can be used to isolate introns and regulatory regions important for expression in the body.

Portions of the disclosed sequence may also be used in polymerase chain reactions as primers. For example, primers can be used to amplify the NOS gene to determine if a mutation is present. The polymerase chain reaction is well known in the art.

Examples

Example 1

This example describes the method by which NOS activity was assayed.

NO synthase activity was measured by monitoring the conversion of [.sup.3 H]arginine to [.sup.3 H]citrulline. For routine assays, we added 25 .mu.l of enzyme extract and 25 .mu.l of 100 nM [.sup.3 H]arginine to 100 .mu.l of buffer containing 50 mM Hepes (pH 7.4), 1 mM NADPH, 1 mM EDTA, 1.25 mM CaCl.sub.2, 1mM dithiothreitol, and 10 .mu.g of calmodulin per ml. After incubation for 5 min at 22.degree. C., assays were terminated with 2 ml of 20 mM Hepes, pH 5.5/2 mM EDTA, and were applied to 1-ml columns of Dowex AG502X-8 (Na.sup.+ form), which were eluted with 2 ml of water. [.sup.3 H]Citrulline was quantified by liquid scintillation spectroscopy of the 4-ml flow-through.

Example 2

This example describes the purification of NO synthase.

Eighteen rat cerebella were homogenized in 100 ml of ice-cold buffer A [50 mM Tris-HCl, pH 7.4/1 mM EDTA/antipain (10 mg/liter)/leupeptin (10 mg/liter)/soybean trypsin inhibitor (10 mg/liter)/pepstatin (10 mg/liter)/chymostatin (10 mg/liter)/phenylmethylsulfonyl fluoride (100 mg/liter)], and all subsequent procedures were carried out at 4.degree. C. The homogenate was centrifuged at 20,000.times.g for 15 min, and the supernatant was loaded at 2 ml/min onto a 20-ml column of diethylminoethyl (DEAE) equilibrated with buffer A. The column was washed with 50 ml of buffer A and eluted with a 100-ml linear gradient of 0-400 mM NaCl in buffer A. Fractions (2.5 ml) were assayed for enzyme activity.

Fractions containing the first peak of activity from the DEAE column (see FIG. 1) were pooled and added to 2 ml of 2', 5'-ADP agarose equilibrated in buffer B (10 mM Tris-HCl, pH 7.4/1 mM EDTA/5 mM 2-mercaptoethanol). After a 10-min incubation, the suspension was poured into a fritted column, which was washed with 50 ml of buffer B with 0.5M NaCl and then with 20 ml of buffer B alone. NO synthase was eluted with 8 ml of buffer B containing 10 mM NADPH.

In our preliminary efforts to purify NO synthase, we observed that enzymatic activity adheres to a DEAE column and can be eluted by 1M NaCl. However, with gradient elution of NaCl, enzymatic activity was not recovered in eluate fractions, suggesting the separation during purification of the enzyme from an important cofactor. Since NO formation requires Ca.sup.2+, we speculated that calmodulin might be involved. Addition of calmodulin to DEAE eluate fractions restores enzyme activity. NO synthase activity elutes in one sharp, major peak followed by a smaller peak of activity, which is observed reproducibly in multiple experiments.

For purification of NO synthase, we have focused on the first, major peak of enzyme activity eluting from the DEAE column, which provides a 5.6-fold purification of enzyme activity with 60% recovery (Table 1, FIG. 1). Further purification utilized affinity chromatography with a 2', 5'-ADP-linked agarose column. NO synthase activity adheres to this column and is not eluted by 0.5M NaCl. After the 0.5M NaCl wash, NO synthase activity can be eluted with 10 mM NADPH, providing a 1000-fold purification of enzyme activity in this step. The overall purification of NO synthase utilizing two steps, DEAE chromatography and 2',5'-ADP affinity chromatography, affords a 6000-fold purification of enzyme activity with 30% recovery. The purified enzyme eluting from the ADP affinity column appears homogeneous, constituting a single band on SDS/PAGE (FIG. 3). The molecular mass of this band is .perspectiveto.150 kDa. To estimate the molecular mass of the native enzyme, we conducted gel filtration chromatography with a Superose-6 column. NO synthase activity of the purified enzyme emerges from the column as a single peak coincident with the peak of protein, with an apparent molecular mass of 200 kDA, similar to the elution of .beta.-amylase whose molecular mass is 200 kDA. Thus, purified NO synthase appears to be a monomer.

Example 3

This example shows the effects of varying concentrations of calcium, calmodulin and NADPH on NO synthase activity.

Calmodulin is an extremely potent stimulator of NO synthase activity (FIG. 2A). In the presence of 1 .mu.M Ca.sup.2+, 50% of maximal stimulation of enzyme activity is apparent with .perspectiveto.200 nM Ca.sup.2+ with maximal enhancement of activity observed at 1 .mu.M Ca.sup.2+ and some reduction in activity at concentrations exceeding 100 .mu.M Ca.sup.2+. In the absence of NADPH, Ca.sup.2+ fails to stimulate NO synthase activity.

Example 4

This example demonstrates the effect of calmodulin antagonists on NO synthase activity.

In crude cerebellar supernatant preparations, calmodulin is not required to demonstrate enzyme activity and added calmodulin (1 .mu.M) has no influence on enzyme activity. However, trifluoperazine, a calmodulin antagonist, inhibits enzyme activity of crude preparations with an IC.sub.50 for trifluoperazine in crude supernatant preparations of vascular endothelial tissue, indicating that regulation of the endothelial and brain enzymes by calmodulin is similar. Trifluoperazine exerts multiple effects such as blockade of dopamine receptors. The drugs W-5 [N-6-aminohexyl)-1-naphthalenesulfonamide] and W-13 [N-4-aminobutyl)-5-chloro-2-naphthalenesulfonamide] are more selective calmodulin antagonists. In crude brain supernatant preparations, W-5 and W-13 inhibit NO synthase activity with respective IC.sub.50 values of 50 and 25 .mu.M.

Example 5

This example demonstrates the properties of purified NO synthase.

The purified enzyme has high affinity for arginine with a K.sub.m of .perspectiveto.2 .mu.M, similar to what we observed previously in crude supernatant preparations. The V.sub.max of the purified enzyme is .perspectiveto.1 .mu.mol per mg of protein per min, similar to the V.sub.max values for other NADPH-requiring oxidative enzymes (Table 2). The K.sub.i for MeArg inhibition of NO synthase activity in the purified enzyme is .perspectiveto.1.4 .mu.M, similar to values we observed previously in crude preparations. The EC.sub.50 for calmodulin enhancement of enzyme activity in the pure enzyme, 10 nM, is similar to the value observed in crude preparations. Also, the EC.sub.50 for calcium stimulation of the purified enzyme is the same in the pure and crude preparations.

The purified enzyme is unstable. When stored at 0.degree. C., 50% of the enzyme activity is lost in 2 hr, whereas the crude supernatant preparation loses 50% activity at 0.degree. C. in 2 days. Stability is enhanced by storing the enzyme in bovine serum albumin (1 mg/ml)/20% (vol/vol) glycerol at -70.degree. C. When stored in this way, the enzyme loses<50% activity in 7 days.

Example 6

This example demonstrates the production and use of antibodies which are immunospecific for calmodulin-dependent NO synthase.

Antibodies were raised in two rabbits and affinity purified with purified NOS. Antiserum was incubated with 50 mg purified antigen (immobilized in nitrocellulose after transfer from an SDS-polyacrylmide gel), eluted with 4M MgCl in 200 mM Tris-HCl (pH 7.4) buffer, and dialyzed against phosphate buffered saline with 0.1% TritonX100.

To ensure that the antiserum interacts with catalytically active NO synthase (NOS, EC1.14.23), we conducted immunoprecipitation experiments. The antiserum precipitates NOS activity in cerebellar homogenates, whether measured by the conversion of arginine to citrulline or by the formation of NO, with half-maximal precipitation at 10 .mu.g ml.sup.-1 antiserum IgG. In Western blot analysis the antiserum interacts with a single band of relative molecular mass 150,000 (M.sub.r 150K), the same as purified NOS. The density of the band varies amongst various brain regions and subdivisions of pituitary and adrenal glands in close parallel with the regional distribution of NOS catalytic activity. Antisera from two rabbits and affinity-purified NOS antibodies provide identical distributions by western blot analysis and by immunohistochemical staining. Immunoreactivity is absent with pre-immune serum or with serum preabsorbed with purified NOS.

Example 7

This example demonstrates the molecular cloning of NO synthase coding sequences.

The nitric oxide (NO) synthase enzyme was purified to homogeneity as described in Example 1. The purified enzyme was run on an SDS gel and transferred to nitrocellulose. Trypsin was added to the nitrocellulose paper containing the enzyme in order to liberate peptide fragments. The peptide fragments were purified by reverse phase HPLC. The peptides were sequenced with an automated peptide sequencer. About 15 peptides were sequenced.

The above procedures were performed in order to obtain peptides whose sequence could be used as a basis for obtaining oligonucleotide probes for molecular cloning. In molecular cloning one frequently prepares a degenerate oligonucleotide based on the amino acid sequence of peptide fragments. For many proteins a mixture of degenerate oligonucleotide probes contains enough correct nucleotide sequence so that hybridization with the cDNA representing the protein to be cloned is possible. When such procedures were carried out for NO synthase, no appropriate clones could be identified. Accordingly, a new technique was developed in which a non-degenerate oligonucleotide probe was generated by polymerase chain reaction (PCR). This was done by taking two of the longest peptides, of 18 and 17 amino acids, and constructing degenerate oligonucleotide primers of 21 nucleotides based on the 7 amino acids at the carboxyl and amino termini of each of the two peptides. These oligonucleotides were employed in a PCR reaction to construct two non-degenerate oligonucleotide primers.

These two non-degenerate oligonucleotide primers were employed in a further PCR reaction to obtain a large polynucleotide probe. There was no way of knowing a priori whether one would obtain an appropriate probe, as the two oligonucleotide primers employed might have been located too far apart in the sequence of the NO synthase to permit amplification. Fortunately, we were able to obtain a 600 base pair amplified product for use as a polynucleotide probe to screen molecular clones.

The 600 bp polynucleotide probe was random prime labeled with .sup.32 P-ATP and used to screen a commercially obtained rat brain cDNA library from Stratagene. Eight overlapping independent clones were isolated and sequenced by double-stranded dideoxy sequencing. This procedure revealed a 4 kb open reading frame coding for a protein of about 150 kD, which corresponds to the molecular weight of NO synthase which we had previously purified.

The deduced amino acid sequence has been examined by computer program for homology with other known families of proteins. No major homology has been observed. A flavin binding site consensus sequence has been observed.

Claims

We claim:

1. A cDNA molecule which encodes all or a portion of a mammalian, calmodulin-dependent Nitric Oxide Synthase (NOS), said molecule comprising between about 12 nucleotides and 4,000 nucleotides.

2. The cDNA molecule of claim 1 which is labeled with a detectable moiety.

3. The cDNA molecule of claim 1 which encodes all of NO Synthase.

4. The cDNA molecule of claim 1 which has the nucleotide sequence shown in SEQ ID NO: 1.

5. The cDNA molecule of claim 1 which encodes the amino acid sequence shown in SEQ ID NO: 2.

6. The cDNA molecule of claim 1 which hybridizes to a molecule having the nucleotide sequence shown in SEQ ID NO: 1.

7. A cDNA molecule which encodes all or a portion of a calmodulin-dependent NOS, and hybridizes to a molecule having the nucleotide sequence shown in SEQ ID NO: 1, said molecule comprising between about 12 nucleotides and 4,000 nucleotides.

8. A recombinant host cell comprising the cDNA molecule of claim 1.

9. The recombinant host cell of claim 8 wherein said cDNA molecule encodes all of the Nitric Oxide Synthase.

10. The recombinant host cell of claim 8 wherein said cDNA molecule has the nucleotide sequence shown in SEQ ID NO: 1.

11. The recombinant host cell of claim 8 wherein said cDNA molecule hybridizes to a molecule having the nucleotide sequence shown in SEQ ID NO: 1.

Non-Patent Literature (20)

Deryak, R., et al., 1984, Cell 38:287-297.
Bredt, D. S., et al., 1990, Society for Neuroscience Abstracts, 16(2): [20th Annual Meeting of the Society for Neuroscience] p. 967, No. 400.10.
Moyer, B., et al., 1990, FEBS Letters, 277(1,2): 215-219.
Knowles, R. G., et al., 1990, in Nitric Oxide from L-Arginine: A Bioregulatory System, Moncada, S., et al. Eds., pp. 139-146.
Knowles, R. G., et al., 1990, Biochemical Journal 269:207-210.
Knowles, R. G., et al. 1990, Biochemical Journal 270: 833-836.
Murphy, M., 1990, Biochemical Journal 271: 563-564.
Hewick, R. M., et al., 1981, The Journal of Biological Chemistry 256(15): 7990-7997.
Lathe, R., 1985, Journal of Molecular Biology 183:1-12.
Frohman, M. A., et al., 1988, Proceedings, National Academy of Sciences, USA 85: 8998-9002.
Kosuga, K., et al., 1990, Biochemical and Biophysical Research Communications, 172 (2): 705-708.
Palacios, M., et al., 1989, Biochemical and Biophysical Research Communications 165(2):802-809.
Rees, D. D., et al., 1990, Biochemical and Biophysical Research Communications, 173(2):541-547.
Kwon, N. S., Nathan, C. F. & Stuehr, D. J., (1989) The Journal of Biological Chemistry, 264:20496-20501.
Furchgott, R. F. & Vanhoutte, P. M. (1988) FASEB J. 3:2007-2018.
Tayeh, M. A. & Marletta, M. A., (1989) The Journal of Biological Chemistry 264:19654-19658.
Bredt, D. S., Hwang, P. M. & Snyder, S. H., (1990) Nature, 347:768-770.
Bredt, D. S., & Snyder, S. H., (1990) Proc. Natl. Acad. Sci. USA, 87:682-685.
Bredt, D. S., & Snyder, S. H., (1989) Proc. Natl. Acad. Sci. USA, 86:9030-9033.
Ignarro, L. J., (1990) Annu Rev. Pharmacol. Toxicol., 30:535-560.

Abstract

Description