Divalent cation-dependent stereospecificity of adenosine 5'-O-(2-thiotriphosphate) in the hexokinase and pyruvate kinase reactions. The absolute stereochemistry of the diastereoisomers of adenosine 5'-O-(2-thiotriphosphate)

31P NMR studies with Cd(II) and Zn(II) chelates of adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and the Cd(II) chelate of adenosine 5'-O-(2-thiotriphosphate) (ATPbetaS) indicate that these metal ions chelate to the sulfur atom of the thiophosphate group. Since Mg(II) chelates to oxygen of the thiophosphate group of diastereoisomer is equivalent to the configuration of the Cd(II) chelate of the opposite diastereoisomer. As a consequence, an inversion of the stereospecificity is observed when Cd(II) is substituted for Mg(II) in the phosphoryl transfer reactions catalyzed by yeast hexokinase and rabbit muscle pyruvate kinase. When Co(II) is the activating ion for yeast hexokinase with ATPbetaS as substrate, no stereospecificity is observed. Since the absolute configuration for the diastereoisomer of Co(III)(NH3)4ATP which is the active substrate for yeast hexokinase has been established by Cornelius and Cleland (Cornelius, R. D., and Cleland, W. W. (1978) Biochemistry, in press), the absolute stereochemistry of the Mg(II) complex of the B isomer of ATPbetaS is now established by its stereospecificity in the hexokinase reaction.     

Use of phosphorus-31 nuclear magnetic resonance to distinguish bridge and nonbridge oxygens of oxygen-17-enriched nucleoside triphosphates. Stereochemistry of acetate activation by acetyl coenzyme A synthetase.

Adenosine 5'-(thiophosphate) AMPS) contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions. A general method was developed to distinguish between the "pro-R" and "pro-S" oxygens. When we converted the AMPS to the isomer A of adenosine 5'-(1-thiotriphosphate) (ATPalphaS), which is known to have S configuration at Palpha, the pro-R oxygen is incorporated into the bridge position, whereas the pro-S oxygen is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge oxygens based on the decrease in the peak intensity of 31P NMR signals caused by the directly bound 17O isotope. The method was used to elucidate the stereochemical course of acetate activation catalyzed by yeast acetyl coenzyme A (CoA) synthetase. The results indicate that yeast acetyl-CoA synthetase is specific for the isomer B of ATPalphaS and that the nucleophilic displacement proceeds with net inversion of configuration at Palpha of ATPalphaS (B), supporting the "in-line" mechanism.     

Carbamyl phosphate synthetase of Escherichia coli uses the same diastereomer of adenosine-5'-[2-thiotriphosphate] at both ATP sites.

Carbamyl phosphate synthetase from Escherichia coli has been shown to use only the A isomer of adenosine-5'-[2-thiotriphosphate] in both the ATPase reaction (MgATP HCO3- leads to MgADP + Pi) and the carbamyl phosphate synthesis reaction (2MgATP + HCO3- + L-glutamine leads to 2MgADP + Pi + carbamyl-P + L-glutamate). The B isomer was less than 5% as reactive. In the reverse reaction, only the A isomer of adenosine-5'-[2-thiotriphosphate] is synthesized from adenosine-5'-[2-thiodiphosphate] and carbamyl-P as determined by 31P NMR and a coupled enzymatic assay with Cd2+- hexokinase. It is therefore proposed that carbamyl phosphate synthetase uses the same diastereomer of MgATP at both ATP sites.     

Investigation of the preferred Mg(II)-adenine-nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP

In the presence of Mg2+ the ecto-(nucleoside diphosphatase) on intact vascular endothelial or smooth muscle cells in culture selectively catabolizes the PS diastereoisomer of adenosine 5'-[alpha-thio]diphosphate, (PS)-ADP [alpha S], and the ecto-(nucleoside triphosphatase) selectively catabolizes the PS isomer of adenosine 5'-[beta-thio]triphosphate, (PR)-ATP[beta S], but exhibits no selectivity towards ATP[alpha S] isomers. In the presence of Cd2+ selectivity to ADP[alpha S] and to ATP[beta S] isomers is reversed; in the presence of Co2+, selectivity is lost. We conclude that each enzyme preferentially recognises the lambda (screw-sense) bidentate Mg(II)-nucleotide complex at its active site.     

On the stereochemistry of activation of phenylalanine by phenylalanyl-tRNA synthetase from baker's yeast.

Because of its chiralic alpha-phosphorus atom adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS) exists in two diastereomeric forms, arbitrarily named (A) and (B). For phenylalanyl-tRNA synthetase ATPalphaS (A) is a substrate whereas ATPalphaS (B) is neither a substrate nor an inhibitor. During the ATPalphaS (A)/PPi exchange reaction with phenylalanyl-tRNA synthetase the configuration at the alpha-phosphorus is retained. The mechanistic implications of these findings are discussed. Preliminary investigations with several other aminoacyl-tRNA synthetases show that the stereochemical requirement with respect to the alpha-phosphorus of ATP is not identical for all aminoacyl-tRNA synthetases.     

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.     

Synthesis of P1,P4-di(adenosine 5'-) tetraphosphate by leucyl-tRNA synthetase, coupled with ATP regeneration

A simple and practical procedure for the synthesis of P1,P4-di(adenosine 5'-) tetraphosphate from ATP by the catalysis of leucyl-tRNA synthetase from Bacillus stearothermophilus is described. Km for leucine was 6.7 microM and for ATP was 3.3 mM. The reaction yielded not only diadenosine tetraphosphate, but various byproducts such as P1,P3-(diadenosine 5'-) triphosphate, ADP and AMP. By coupling the reaction with an ATP regeneration system by acetate kinase and adenylate kinase with acetylphosphate as a phosphate donor, diadenosine tetraphosphate was prepared as a sole product at a high yield (96%).     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P nuclear magnetic resonance spectra of the thiophosphate analogues of adenine nucleotides; effects of pH and Mg2+ binding.

The 31P nuclear magnetic resonance (NMR) spectra of the adenine nucleotide thio analogues, AMPS, ADPalphaS, ADPbetaS, ATPalphaS, ATPbetaS, and ATPgammaS, have been studied. Of primary interest were the increased sensitivity of chemical shifts to protonation and to magnesium binding of these analogues compared with the corresponding effects on AMP, ADP, and ATP. The usefulness of the characteristic NMR parameters of the thio analogues as probes in enzymatic reactions is discussed. The A2 diastereoisomers of ADPalphaS and ATPalphaS and the A and B isomers of ATPbetaS were enzymatically synthesized and the diasterioisomers of ADPalphaS and ATPbetaS were distinguished by their 31P NMR parameters. The stereospecificity of the enzymatic reactions involving the thio analogues of nucleotides can therefore be determined by 31P NMR. The difficulty involved in assigning phosphate ligands of Mg in MgADP and MgATP and their analogues on the basis of the magnitude of chemical shift changes (deltadelta) induced by Mg binding upon each 31P is discussed in the context of the anomalies in deltadelta of each 31P observed upon protonation of the terminal phosphate group. It is concluded that chemical shift data cannot yield unequivocal information concerning the absolute structure of metal complexes of nucleotides but can be used to monitor changes in metal chelation, for example, upon binding to enzyme.     

A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.

A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.     

Structure of the metal-nucleotide complex in the acetate kinase reaction. A study with gamma-32P-labeled phosphorothioate analogs of ATP

The synthesis of the gamma-32P-labeled diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and the Sp isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) by a modification of the Glynn and Chappell method (Glynn, I. M., and Chappell, J. T., (1964) Biochem. J. 90, 147-149) is described. These analogs were tested as substrates for acetate kinase in the presence of several divalent metal ions. Both isomers of ATP alpha S are substrates in the presence of Mg2+, Mn2+, Co2+, Zn2+, and Cd2+, the Sp isomer being preferred by a factor of between 4.8 (Mg2+) and 52.5 (Cd2+). Only the Rp isomer of ATP beta S is a substrate in the presence of Mg2+, and the Sp isomer becomes a better substrate in the presence of Mn2+, Co2+, and Zn2+; both isomers are equally good substrates in the presence of Cd2+. The change in specificity upon replacing Mg2+ by Cd2+ is greater than 1800 at beta-phosphorus and 10 at alpha phosphorus. These results provide a basis for proposing that the lambda screw sense configuration of the beta, gamma-bidentate MgATP complex is the substrate for acetate kinase. In the reverse reaction, both Sp and Rp isomers of ADP alpha S are substrates in the presence of all metal ions tested, the Sp isomer preferred by a factor between 12.3 (Mg2+) and 45.5 (Cd2+). In the presence of Mg2+, Mn2+, and Co2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S, while a mixture of Rp and Sp isomers is synthesized in the presence of Zn2+ and Cd2+. These results are analogous to those for the forward reaction and suggest that the Mg.ADP complex which binds as a substrate in the reverse reaction, and is released as a product in the forward reaction, is the beta-monodentate. The classification of acetate kinase as an enzyme having a type I mechanism (Dunaway-Mariano, D. and Cleland, W. W. (1980) Biochemistry 19, 1506-1515) for kinases, is discussed.     

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.     

Interaction of the recA protein of Escherichia coli with adenosine 5'-O-(3-thiotriphosphate)

Incubation of the recA protein of Escherichia coli with the ATP analog adenosine 5'-O-(3-thiotriphosphate) (ATP(gamma S)) in the presence of DNA produces an irreversible inhibition of ATPase activity, although in the presence of ATP, ATP(gamma S) shows an initial competitive inhibition. ATP(gamma S) is not appreciably hydrolyzed by recA protein and the inhibition of ATPase activity is due to the formation of stable complexes which contain equimolar amounts of ATP(gamma S) and recA protein. Formation of stable complexes requires DNA, which is also stably bound to recA protein in the presence of ATP(gammaS), at a ratio of 5 to 10 nucleotides/recA protein monomer. The DNA requirement is satisfied by either single-or double-stranded DNA, and in the latter case, the pH dependence is comparable to that observed for ATP hydrolysis. Binding of ATP(gamma S) is inhibited by other nucleoside di- and triphosphates with efficiencies corresponding to their inhibitory effects on the ATPase activity of recA protein.     

Structural and biochemical properties of bidentate tetraaquarhodium(III) complexes of inorganic pyrophosphate and adenosine 5'-diphosphate

The structural and biochemical properties of the alpha,beta-bidentate tetraaquarhodium(III) complexes of inorganic pyrophosphate [Rh(H2O)4PP] and adenosine diphosphate [Rh(H2O)4ADP] are examined. These Rh(III) complexes are exchange-inert analogues of the corresponding physiologically important MgIIPP and MgIIADP complexes. The crystal structure of [Rh(H2O)4H2P2O7]+Cl- shows that the six-membered chelate ring adopts a twist-boat conformation with an unusually high puckering amplitude of 0.756 (3) A. The Rh coordination distances average 2.02 (1) A, while the bridge P-O bonds are virtually equal in length. All 10 protons of the complex participate in hydrogen bonding. There are two intramolecular hydrogen bonds between the phosphate oxygen atoms and the axially coordinated water molecules. The Rh(H2O)4PP complex was found to be a substrate for yeast inorganic pyrophosphatase, with Ki = 0.063 (7) mM and Vm = 500 (100) min-1. The two screw sense isomers of Rh(H2O)4ADP were prepared from (Rp)-[alpha-16O,18O]ADP and assigned configuration on the basis of the magnitude of their 31P NMR isotopic chemical shifts. The Rh(H2O)4ADP complex binds a number of kinases as tightly as MgADP. Arginine kinase and creatine kinase were shown to bind the delta Rh(H2O)4ADP isomer 7 and 45 times tighter, respectively, than the lambda isomer. The reactivity of Rh(H2O)4PP with pyrophosphatase is comparable to that of Cr(H2O)4PP, and the binding affinities of the Rh(H2O)4ADP screw sense isomers for kinases are also comparable to those observed for the corresponding Cr(H2O)4ADP screw sense isomers.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate)

Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.     

Identification of tyrosine and lysine peptides labeled by 5'-p-fluorosulfonylbenzoyl adenosine in the active site of pyruvate kinase

The nucleotide affinity label 5'-p-fluorosulfonylbenzoyl adenosine reacts at the active site of rabbit muscle pyruvate kinase, with irreversible inactivation occurring concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit (Annamalai, A. E., and Colman, R. F. (1981) J. Biol. Chem. 256, 10276-10283). Purified peptides have now been isolated from 70% inactivated enzyme containing 0.7 mol of reagent/mol of enzyme subunit. Rabbit muscle enzyme labeled with radioactive 5'-p-fluorosulfonylbenzoyl adenosine was digested with thermolysin. Nucleosidyl peptides were purified by chromatography on phenylboronate-agarose and reverse-phase high performance liquid chromatography. After amino acid and N-terminal analysis, the peptides were identified by comparison with the primary sequences of chicken and cat muscle enzyme. About 75% of the reagent incorporated was distributed equally among three O-(4-carboxybenzenesulfonyl)tyrosine-containing peptides: Leu-Asp-CBS-Tyr-Lys-Asn, Val-CBS-Tyr, and Leu-Asp-Asn-Ala-CBS-Tyr. These tyrosines are located in a 28-residue segment of the 530-amino acid sequence. The remainder of the incorporation was found in two N epsilon-(4-carboxybenzenesulfonyl)lysine-containing peptides. Leu-CBS-Lys and Ala-CBS-Lys-Gly-Asp-Tyr-Pro. Modification in the presence of MnATP or MnADP resulted in a marked decrease in labeling of these peptides in proportion to the decreased inactivation. It is suggested that these modified residues are located in the region of the catalytically functional nucleotide binding site of pyruvate kinase.     

Adenosine 5'-(beta, gamma-imino)triphosphate and guanosine 5'-O-(2-thiodiphosphate) do not necessarily provide non-phosphorylating conditions in adenylate cyclase studies

Commercial preparations of adenosine 5'-(beta, gamma-imino)triphosphate (App(NH)p) were found to be contaminated with a GTP-like substance(s) as well as a phosphate donor(s) for GDP. Thus, when these preparations were used as substrate with no purification, GDP was as effective as GTP in promoting PGE1 stimulation of human platelet adenylate cyclase. With purified App(NH)p as substrate, the effect of PGE1 with GDP was reduced but still observable, while that with GTP was unaltered. PGE1 also caused a stimulation in the presence of guanosine 5'-o-(2-thiodiphosphate)(GDP beta S) with ATP as substrate. Both of the PGE1-stimulated activities observed with GDP and its analog were completely lost by the addition of UDP, thereby, inhibiting GTP formation catalyzed by membrane-associated nucleoside diphosphate kinase. The results demonstrate that the stimulatory effects of PGE1 observed with GDP and App(NH)p, and with GDP beta S and ATP were transphosphorylation dependent and, therefore, the analogs must be used with special caution in adenylate cyclase studies.     

Irreversible inhibition of adenylate cyclase by 5'-(p-bromomethylbenzoyl) adenosine

The effect of 5'-(p-bromomethylbenzoyl) adenosine (pBMBA) on adenylate cyclase from bovine caudate nucleus membranes was studied. Adenylyl-5'-methylenediphosphonate (but not adenosine) protected adenylate cyclase against inactivation by this compound. The degree of pBMBA-induced inhibition of adenylate cyclase increased in the presence of Mg2+. 5'-(p-fluorosulfonylbenzoyl) adenosine (pFSBA) was also a specific irreversible inhibitor of adenylate cyclase. It was demonstrated that the enzyme inactivated by pFSBA completely restored its activity under the action of dithiothreitol. The results obtained are indicative of the presence of the -SH group in the enzyme active site.