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Supplement F2: The Chemistry of Amino, Nitroso, Nitro and Related Groups.

Edited by Saul Patai Copyright 1996 John Wiley & Sons, Ltd.

ISBN: 0-471-95171-4

CHAPTER 22

 

Nitric oxide from arginine:

 

a biological surprise

 

ALAN H. MEHLER

 

Department of Biochemistry and Molecular Biology, Howard University,

 

Washington, DC, USA

 

I. ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

974

II. ABBREVIATIONS AND DESIGNATIONS USED

 

IN BIOCHEMISTRY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

974

III. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

974

A. Early Reports on NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

974

B. Mechanism of NO Formation . . . . . . . . . . . . . . . . . . . . . . . . . . .

975

C. Content of Folate and the Origin of Oxygen . . . . . . . . . . . . . . . . .

976

IV. INDIVIDUAL NOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

976

A. bNOS cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

976

B. eNOS cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

977

C. iNOS cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

978

V. LOCALIZATION OF THE HUMAN GENE . . . . . . . . . . . . . . . . . . .

978

A. bNOS in the Human Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

978

B. eNOS in the Human Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

979

C. iNOS in the Human Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

979

D. Three Genes come from Three Chromosomes . . . . . . . . . . . . . . . .

980

VI. FORMATION OF NO AND CITRULLINE . . . . . . . . . . . . . . . . . . . .

980

VII. COMPLEXITIES OF NOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

983

A. Role of BH4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

983

B. Iron in NOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

983

C. Subunits and Dimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

987

D. Complications of Many Cofactors . . . . . . . . . . . . . . . . . . . . . . . .

989

E. Inhibitors of NOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

990

F. Factors for Growth of NOS . . . . . . . . . . . . . . . . . . . . . . . . . . . .

991

VIII. PHYSIOLOGICAL FUNCTIONS OF NOS . . . . . . . . . . . . . . . . . . . .

992

IX. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

993

X. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

993

973

974

Alan H. Mehler

 

I. ABBREVIATIONS

bNOS

brain nitric oxide synthase

BH4

tetrahydrobiopterin, tetrahydrofolate

cDNA

complementary DNA

cGMP

30 ,50 -cyclic phosphate of guanylic acid

EDRF

endothelial-derived relaxing factor

eNOS

endothelium nitric oxide synthase

FAD

flavin adenine dinucleotide

FMN

flavin mononucleotide

iNOS

inducible nitric oxide synthase

NADPH

nicotinamide adenine diphosphate phosphate reduced

L-NAME

NG-nitro-L-arginine methyl ester

NO

nitric oxide

NOS

nitric oxide synthase

II. ABBREVIATIONS AND DESIGNATIONS USED IN BIOCHEMISTRY

Biochemists started more than 50 years ago to name familiar molecules by abbreviations. They have not been dissuaded by the change in terminology nor the alterations in names. Therefore, the current list of short-hand abbreviations that are used now takes several pages. The sort of names used in this chapter is quite varied and is illustrated below.

For distance along nucleotide length, the dimension is kilobases, kb. The nomenclature for the weight of proteins is one of several possibilities. The one used is Kilodaltons, Kd. The position of genes upon a chromosome is given by either p, for the shorter portion from the centromere, or q, for the longer portion. Within the p or q, the distance is given as staining bands with the Giemsa stain, as for example 7q35 7q36 represents the 7th chromosome, position 35 36 of the distance away from the centrosome. The designation qter represents the terminal portion of the chromosome.

DEAE is the abbreviation for DiEthyl Amino Ethyl, the binding group of a substitution on cellulose. SDS/PAGE is a technique for naming the material used for electophoresis in which SDS represents sodium dodecyl sulfate solution that is used with PolyAcrylimide Gel Electrophoresis (PAGE).

TATA and CCAAT arc the names where, in general, the polymerase binds to DNA, but these factors are sometimes missing. The other binding sites for the many factors that activate the polymerase, Sp1, AP-1, etc., have many diverse meanings. SOD is superoxide dismutase. The abbreviations as part of the names such as NG- or Nω - for the L-NAME indicate the nitrogen atoms listed as either the guanido nitrogen (G) or the terminal nitrogen ω (two ways for listing the same nitrogen atoms).

III. INTRODUCTION

A. Early Reports on NO

The discovery that a biological catalyst was nitric oxide (NO) was a surprising event. This finding was in part due to the persistence of Furchgott1, who discovered in 1987 that what he called EDRF (endothelium-derived relaxing factor) was probably this molecule and Ignarro2, who determined in the same year that EDRF and NO had the same halflife and were released in the same order by various treatments of arterial or venous substrates; cGMP was induced and the materials were bound by hemoglobin. These two findings were reported in a symposium organized by Vanhoutte3 and were the first findings

22. Nitric oxide from arginine: a biological surprise

975

that a new era was dawning. Inspired by this discovery, Moncada and collaborators in the Wellcome Research Laboratories4 tried a group of physical approaches that also favored the existence of nitric oxide as the material proposed by Furchgott and by Ignarro. It should be emphasized that all of the experiments reported by 1988 were pharmacological and were of primary interest to pharmacologists. However, from that time until the present (1995), many papers have involved biochemists, physicians and other scientists and the number of papers on the subject of nitric oxide has reached over 1000 in 1994.

The first paper by Furchgott on EDRF was published in 19805. This work showed that acetylcholine was effective in producing the effect of a relaxation of rabbit thoracic aorta only if endothelial cells were still present and that these were easily removed. It is necessary to recall that the endothelial cells are only a single layer on the internal surface of a much larger muscle layer. However, the single layer of endothelial cells comprises the essential components for producing the active principle. The production of NO was not completely unexpected. Some 10 years prior to Furchgott and to Ignarro’s discovery, in a review by Murad’s group6, the efficiency of NO was shown upon cGMP and the inhibition by hemoglobin was also demonstrated. However, the technical tools necessary for determining the source and even the identity of this compound were not available. This was true even though Keilin and Hartree had shown that catalase formed NO back in 19547.

While the statements that EDRF was really NO were still in progress, Moncada and collaborators started studies that gave further evidence that the active ingredient was in fact the simple gas. In a series of papers in 1987, Radomski, Palmer and Moncada showed that material released from endothelial cells and NO were similar in their passage through a cascade and decomposed at similar rates8,9. A parallel study showed that platelets10 and vascular smooth muscle were effected in the same way by both EDRF and NO11,12. The activation by superoxide dismutase (SOD) and inhibition by hemoglobin (Hb) were similar for both of these11,12. In these experiments, the concentration of NO was determined by chemiluminescence following the reaction with ozone11.

It should be noted that many of the data of Moncada were confirmations of the data of others. Thus, the work of Furchgott showed that Hb inhibited EDRF action12 16. The laboratory of Murad had very early shown the effects of many nitro compounds, including NO, on the stimulation of cyclic guanylic acid (cGMP)17,18 and Craven and DeRubertis19 had shown that NO along with a number of related compounds was capable of stimulating cGMP and that nitrohemoglobin had a similar effect. Gruetter and coworkers20 and Mellion and collaborators20 22 reported that NO is a good inhibitor of platelet aggregation based on work of Needleman’s group23,24. Ignarro25 28 found NO was liberated from nitrosothiols and activated guanylate cyclase, elevated the vascular and platelet level of cGMP, caused vascular smooth muscle relaxation, inhibition of platelet aggregation and hypotension in anesthetized animals.

B. Mechanism of NO Formation

By the year 1987, there was intense excitement about the biosynthesis of NO. The discovery that the active molecule was derived from arginine was strange enough. This was first shown by Hibbs and coworkers29, who showed that mouse macrophages required L-arginine for cytotoxic activated macrophages (CAM) and also for inhibition of aconitase and uptake of [3]-thymidine into DNA. The reaction was also given by L-homoarginine and some derivatives of L-arginine and N-monomethyl arginine (NGMMA) was particularly inhibitory. Shortly after, the same group30 showed that arginine was converted to citrulline.

976

Alan H. Mehler

Iyengar and coworkers31 found that the oxidation of L-arginine led to citrulline, actually before Hibbs and collaborators30. At this point in time, Palmer’s group11 showed that NO was indeed formed from the guanido nitrogen of arginine. This led to a series of confirmations; the first was Ignarro and collaborators32, who demonstrated that NO was the same as EDRF. Then in 1988 Marletta and coworkers33 showed that nitric oxide was formed and that NO2 and NO3 were formed from it. In their reaction L-arginine MgCC and NADPH were required. The enzyme was soluble in mouse macrophage, the RAW 264.7 cells. Later in 1988, Hibbs and collaborators34 showed that mouse macrophages formed NO from arginine and that NO was the precursor of NO3. This was confirmed in 1989 by Stuehr’s group35, who showed that the production of NO from L-arginine was responsible for CAM and that the process was inhibited by NG, NG-dimethyl-L- arginine.

C. Content of Folate and the Origin of Oxygen

The complexity of NO-synthase was emphasized by Tayeh and Marletta36 and Kwon, Nathan and Stuehr37, who isolated dihydroand tetrahydrofolate as the endogenous compounds that stimulated the oxidation from 20 30% to 100%. Kwon and coworkers38 followed this finding with mass spectrometric data, which showed that the oxygen of citrulline formed from arginine contains the oxygen of air. Leone’s group39 demonstrated that both the citrulline and NO oxygen are derived from molecular O2 by showing that both methylate citrulline and nitrosomorpholine contained the isotope of the oxygen gas. This was found to be true for the three types of NO synthetase (see below) except for the NO from brain, which was not recovered as a morpholine derivative.

IV. INDIVIDUAL NOS

A. bNOS cDNA

The presence of NO in mammalian brain was first proposed by pharmacologists. Using cerebellar cells, Garthwaite and collaborators40 determined that EDRF was produced by N-methyl-D-aspartate (NMDA) and that NO was the probable nature of the EDRF. While this work was under way, Knowles and coworkers41 demonstrated the synthesis of NO and citrulline in a crude synaptosomal cytosol from rat forebrains. This work established that NADPH was required, that a number of simple arginine derivatives were effective, that hemoglobin was inhibitory and that L-methylarginine prevented the activity. Within months of the work of Knowles, Bredt and Snyder42 purified an enzyme from cerebella of 10-day-old rats that synthesized NO. The technique involved the formation of citrulline, since this compound can be easily determined. This work was continued and Bredt and Snyder43 within a few months of their previous study had purified the brain NOS. This work was dependent upon a couple of factors: first, the requirement of calmodulin for the enzyme and, second, the elution of the enzyme from 20 ,50 -ADP agarose with NADPH. The use of calmodulin is important for the role of calcium as an activator of the enzyme. The first elution gave a six-fold purification on DEAE. The second step, 20 ,50 -ADP agarose, allowed the bulk of the protein to be eliminated with 0.5 M NaCl followed by elimination of NO synthase with 10 mM NADPH.

The isolation of NOS led to the cloning and expression of the cDNA for this enzyme. NOS was purified as before and tryptic fragments were isolated. Bredt’s group44 then continued to isolate the cDNA by a complex method. The two larger peptides (17 and 18 amino acids) were formed, then a 599-bp product was synthesized from the two of these. This 599-bp product was then used to screen 106 cDNA clones and three clones

22. Nitric oxide from arginine: a biological surprise

977

had an open reading frame of 4,287 bases. This corresponded to a molecular mass of about ¾160K and incorporated all of the 21 peptides given by trypsin.

The cDNA was inserted into human kidney cells with cytomegalus virus and gave a single Coomassie blue stain, which produced citrulline from arginine, produced nitrite from arginine and produced NO, which gave increased endogenous cGMP synthesis. The cDNA showed a 10.5 Kb band in total cerebellar RNA, which indicates that over half the mRNA is not expressed. NOS RNA was not expressed in kidney, liver, skeletal muscle, stomach or heart. The largest amounts were present in cerebellum, then less so in olfactory bulb, colleculi, hypocampus and cerebral cortex. Comparing the structure with those in Gene Bank, this was found to resemble cytochrome P-450. The amino acids from the C-terminal portion, 641 amino acids, have a homology with the cytochrome P450; there is a 36% identity and 58% close homology. The fact that both enzymes use both FMN and FAD suggests that there is a mechanism for transferring between them.

This finding was confirmed by Mayer and coworkers45. Using pork cerebellum, they employed similar purification to obtain a 45-fold enzyme. The factors responsible for activation were the same as those for rat: NADPH, CaCC plus calmodulin, FAD and, in an unknown way, BH4. The molecular weight was also 160,000 kDa. A similar purification from rat cerebellum was made by Schmidt and collaborators46, who also obtained similar values although they claimed four major factors were given by SDS/PAGE. The difference might have been attributable to the use of generic cerebella by the latter workers.

B. eNOS cDNA

The success of Bredt’s group44 in cloning NOS from cerebellum raised questions about the similar cloning of endothelial cell NOS, largely because these two have roughly identical properties. In 1988, Palmer and Moncada47 and Schmidt and coworkers48 independently found that endothelial cells formed NO from L-arginine. Pollock’s group49 purified the enzyme from bovine aorta. The preparation was 95% insoluble but was converted to a form that was soluble on treatment with 3-[(3-cholamidopropyl) dimethylaminoniol]-propane sulfate (CHAPS). The soluble form with CHAPS was used. The purified enzyme required BH4 for maximum activity, CaCC and calmodulin and NADPH for any activity. Arginine was converted to citrulline, NO3 was created and the preparation had EDRF activity. The molecular weight was 135 kDa.

The cDNA responsible for the aortic protein from bovine source was purified by four groups simultaneously, namely by Janssen’s group50, by Sessa’s group51, by Lamas and coworkers52 and by Nishida’s group53. All four derived the same sequence of bases. Those include methionine, which is in a consensus sequence for initiation54. Also included are a calmodulin site, an FMN site, a pyrophosphate and an FAD site for the dinucleotide and the NADPH sites for ribose and adenine. The three cDNAs made NO according to different assays, including NADPH diaphorase, a cyclic GMP assay, interfered with by NAME, synthesis of citrulline from arginine and NO synthase in COS cells. The fact that this 135 kDa protein was associated with the cell structure was pointed out by the authors as due to a myristyl site, which was not found in the brain cDNA. The report of Nishida showed a sheer stress in the cDNA.

Endothelial NOS was found to be associated with cell membranes and this property was associated with the finding that the cDNA contained the structure for binding myristic acid55. Busconi and Michel56 used bovine aortic endothelial cells to demonstrate that the myristoylated protein was found. When the second amino acid, glycine, was converted to alanine, the addition of myristic acid was prevented and the enzyme remained soluble. Sessa and coworkers51 carried out the same experiments simultaneously and found the same results. However, in both cases the enzyme was not purified, although the protein

978

Alan H. Mehler

was found to include myristic acid. The protein was shown to be modified by Liu and Sessa57. They found the endothelial NOS was indeed myristylated, that the myristic acid was not modified during the binding and that the binding is an amide linkage. Of course, the N-terminal methionine is lost during the preparation of the enzyme for myristoylation.

C. iNOS cDNA

Macrophages were among the first cells found to produce NO29. On purification, the macrophages from various animals were found to be similar to the neural43 and endothelial cells47,49 in that they contained an enzyme that decomposed arginine to citrulline and NO31,39. The enzyme used NADPH for the reaction39; molecular oxygen was the source of the oxygen included in the products38,39 and the enzyme was only partially active in the absence of BH436,37. The major difference between the enzymes was the complete lack of effect of CaCC and the independence of the activity upon calmodulin58,59. Another difference was the inducibility of the macrophage enzyme60 62. Both interferonand lipopolysaccharide were required for maximum expression of activity63 and, contrary to the short half-life of the neuronal enzyme (seconds), the half-life of the macrophage was hours64.

The lack of calmodulin in the macrophage enzyme was explained by the work of Cho and coworkers65, who reported that the purified enzyme contained calmodulin bound through noncovalent bonds. This followed the report by Stuehr’s group58 that the macrophage enzyme was purified from RAW 264.7 cells derived from mice and that this enzyme had, in addition to the features above, both FAD and FMN. This work strengthened the report of Yui and collaborators66, which described the enzyme from rat macrophages but did not find evidence for calmodulin or flavins. A similar report from Hevel and coworkers67 described the purification from mouse macrophages of an enzyme that contained 1 FMN and 1 FAD; this also oxidized arginine to citrulline and NO better in the presence of BH4.

At this stage of discovery, the encouragement was set for cloning of the macrophage enzyme. This was carried out independently by three groups, Lyons and coworkers68, Xie and collaborators63 and Lowenstein’s group69. The first group68 showed the gene contained FMN, FAD and NADPH, the second group63 showed in addition the calmodulin binding site. Lowenstein’s group69 confirmed the findings of Xie and coworkers and showed the picture of spleen cells with both red pulp and, less, in white pulp. All three teams showed the molecular weight of 130,000.

V.LOCALIZATION OF THE HUMAN GENE

A.bNOS in the Human Gene

The localization of bNOS to the human genome was accomplished by Kishimoto and coworkers70. These investigators used a rat cerebellar cDNA to obtain a human cDNA from Clontech. This cDNA was hybridized to Southern blots containing DNA from a battery of human-rodent somatic cell DNA. Since the blots had been shown to be selective, the authors showed that the cDNA hybridized to chromosome 12. By using restriction nucleases EcoRI and Hind III the assignment was made to 12 q14-qter. One or two copies were indicated in vivo but reducing the hybrid conditions showed more bands. It is necessary to conduct further studies to see whether the other cDNA are derived from this or another clone.

A more complete analysis of human neuronal NOS gene was made by Hall’s group71. It is a gene of 29 exons; a flanking region of over 1500 bp was determined 5- and several

22. Nitric oxide from arginine: a biological surprise

979

poly (A) are found as far as nt 6632: A region for heme in exon 6, CaCC /calmodulin in exons 13 and 14, FMN in exon 18, FAD in exons 21, 22 and 23 and NADPH in exons 25, 26 and 27. Although promoters have yet to be determined in vivo, diversity is suggested by the occurrence of AP-2, TEF-1/MCBF, CREB/ATF/cFOS, NRF-1, Ets, NF-1 and NF- B in the 5-region.

B. eNOS in the Human Gene

Janssens and coworkers50 cloned the cDNA for endothelial NOS from human tissues. The cDNA contained the FMN, FAD and NADPH sequences attributed to these cofactors. The enzyme was CaCC -dependent and this was blocked by L-NAME. More than 95% of the enzyme sedimented in the particulate fraction. The enzyme made cGMP in reporter cells, corresponding to NO synthesis, and this was antagonized by L-NAME. These properties of cDNA from human endothelial NOS were basically confirmed with several differences noted by Marsden’s group72 in the process of isolating and characterizing the human gene and locating it in 7q35 7q36. The gene was isolated from human clones in a bacteriophage library. This gene lacks the TATA box but includes a CCAAT box, Sp1 sites, GATA sites and reverse sites; AP-1 site, AP-2 site, physical stress elements and heavy metal sequences were found. Steroid binding sites were lacking. The consensus sequence for RNA polymerase III was found. The chromosome map was determined with human-rodent pairs at 7q32 7qter and was done more accurately with the FISH determination with metaphase chromosomes. This latter analysis showed that the gene was 7q35 36. The same authors also obtained a more precise location of the human bNOS as 12q24.2. This gene also contained the calmodulin/calcium site and the FMN, FAD and NADPH binding sites.

Zhang and coworkers73 confirmed the work in eNOS of Marsden’s group72 with about 10 differences in the 50 -region, none of which was a promoter box. They found the number of promoters and Sp1 and GATA sites were effective. There is a likelihood of several others participating also. Busconi and Michel74 tested eNOS for membrane targeting and found that only the myristyl portion of the molecule, not the polybasic region, is responsible for membrane association. Venema and collaborators75 grew the eNOS in baculovirus. They found less than theoretical FAD and FMN and hope to find conditions for putting these flavins in the enzyme in proper quantities.

C. iNOS in the Human Gene

A human gene for hepatic inducible NOS was isolated in 1993 by Geller and coworkers76. Hepatocytes were isolated from an operative wedge resection, which were over 98% pure. The cells were stimulated with cytokines TNF-˛, IL-1 and IFN- . The purified cDNA, in addition to FMN, FAD and NADPH factors, also contained a calmodulin site. This site retained some activity in the presence of calcium inhibitors. There are also phosphorylation sites at 232, 576 and 890 residues.

In a paper completing the series, Xu’s group77 confirmed the structure of human endothelial NOS as occupying 7q35 36 and found the gene for human inducible NOS as being on chromosome 17. This was a short paper that made the point that each of human genes for NOS, brain, endothelial are inducible, as a separate product of similar but related genes. A more complete paper by Chartrain and coworkers78 isolated the gene from human foreskin fibroblasts. The gene was shown to reside on chromosome 17 cenq11.2. A comparison of hepatic cDNA for NOS with the gene sequence showed 99.7% identity and the 0.3% difference is attributed to polymorphism, since the sources differed.

980

Alan H. Mehler

D. Three Genes come from Three Chromosomes

In the few years since NOS was shown to be an ubiquitous enzyme, the enzyme had been purified, the mRNA was identified and the gene had been isolated and mapped to human chromosomes. The findings were the sort predicted from the beginning: the three types of NOS were indeed different from each other and were represented by three closely related, but distinct, genes occurring on three chromosomes in man. The three genes are large structures and it is possible that many introns will be responsible for types of one or more of the types. It should be noted, however, that only one gene exists of each type.

VI. FORMATION OF NO AND CITRULLINE

The formation of citrulline from arginine led Hibbs and collaborators34 to postulate that the reaction known to occur in microorganisms was also found in eukaryotes. They proposed that the reactions shown in Figure 1 occurred. The reaction was specific for L-arginine and was also given by L-homoarginine but by no other guanidino compounds. A short time later, Iyengar’s group31 showed that the mixture of NO2/NO3 was derived from the guanidino groups of L-arginine and that, since citrulline was probably a product, only a single N contributed. Since NO3 is not derived from NO2, the synthesis of these compounds is conjectural. They wrote the reaction as shown in Figure 2.

Palmer and Moncada79 found a similar reaction in porcine aortic endothelium but were reluctant to designate the same reaction as Hibbs and coworkers39 because they thought there was little point in reducing the nitrogen to ammonia before oxidizing it. They found NNMA to be a powerful inhibitor.

Kwon, Nathan and Stuehr37 found tetrahydrofolic acid (BH4) to serve as a cofactor for the production of NO from mouse macrophages. This compound had been established

 

 

 

 

 

Deminase

 

 

 

 

 

 

L-arginine C H2O ! citrulline C NH3

 

 

 

 

1 1 O2

oxidase

 

 

 

 

HC

 

 

NH3

C

!

NO2

C

H2O

C

 

 

 

2

 

 

 

FIGURE 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+

 

 

 

 

 

H 15N

 

 

 

 

NH3

 

 

 

 

 

2

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

15NO2 + 15NO3

 

C

NCH2 CH2 CH2 CH

 

 

H 15N

 

H

 

COO

 

 

 

 

2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O

O

N

15N

O

N

H

FIGURE 2

22. Nitric oxide from arginine: a biological surprise

981

FIGURE 3

H2 N O

C

NH

NADPH

 

 

NADP+

 

Dihydrofolate

 

reductase

 

 

 

 

 

 

 

 

 

MTX

 

H2 Biopterin

 

 

 

 

NA D(P)H

H4 Biopterin

QH2 Biopterin

 

Dihydropteridine

 

 

 

reductase

NO2

+

NO3

O2

NO

+

LCitrulline

+

H2 N NH2

NH

+

COO

H3 N

L-Arginine

N C

NMA

 

 

 

 

NO generating

 

Enzyme(s)

NADPH(?)

 

 

LArginine

+

H2 N NHOH

NH

+

 

COO

 

H3 N

 

 

L-NHA

 

 

 

 

 

 

 

N

 

O

 

 

 

 

 

 

 

 

 

 

 

 

 

O2

 

 

 

 

H2 O

 

 

NO

 

 

 

NO

2

3

NH

 

 

 

 

 

 

+

H2 N N O

NH

+

 

COO

H3 N

 

NG-oxo-L-arginine

 

 

H

 

 

+

 

 

 

 

H2 N

 

NO

N

+

COO

+

COO

+

COO

H3 N

H3 N

H3 N

L-citrulline

FIGURE 4

982

Alan H. Mehler

+

 

+

 

H2 N

NHCH3

H2 N

N CH3

 

ΝΗ

H+

ΝΗ

 

 

+

COO

+

COO

H3 N

H3 N

L-NMA

 

 

 

FIGURE 5

+OH

H2 N N

CH3

ΝΗ

+

COO

H3 N

+H

H2 N N CH2

ΝΗ

H3 N COO

+

 

OH

 

 

 

H2 N

 

NH CH2

 

ΝΗ

+

COO

H3 N

Carbanolamine

−Η 2Ο

+

H2 N N CH2

ΝΗ

+

COO

Η3Ν

 

imine

Соседние файлы в папке Patai S., Rappoport Z. 1996 The chemistry of functional groups. The chemistry of amino, nitroso, nitro and related groups. Part 2