Interaction of nucleotide affinity analog 5'-p-fluorosulfonylbenzoyladenosine with platelet ADP receptor: aggregin.
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Adenosine↗
Biomedical subjects
R F Colman
Publications and source records attributed to R F Colman.
Clopidogrel inhibits the binding of ADP analogues to the receptor mediating inhibition of platelet adenylate cyclase.
Clopidogrel, like the homologous thienopyridine derivative ticlopidine, selectively inhibits platelet aggregation induced by ADP. We have previously described two nucleotide-binding sites on platelets related to ADP-mediated platelet responses. The first is a high-affinity binding site for 2-methylthio-ADP (2-MeSADP) that is linked to the inhibition of stimulated adenylate cyclase. The second is the 100-kd exofacial membrane protein aggregin, which is labeled by the reactive ADP analogue 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) that is related to shape change and aggregation. We set out to determine if either of these sites is blocked in vivo by clopidogrel or its active metabolite. Six subjects were given clopidogrel (75 mg/day for 10 days) in a double-blind crossover experiment. All of the subjects developed prolonged bleeding times while taking the drug. The rate of onset of the effect on bleeding time varied among subjects. Platelet aggregation induced by ADP or thrombin was significantly impaired by the drug treatment, but no effect was detected on shape change. The incorporation of [3H]FSBA into aggregin was also unaffected. Inhibition of adenylate cyclase by ADP or by 2-MeSADP was greatly reduced in all subjects, and in the case of 2-MeSADP, there was evidence for a noncompetitive effect. Inhibition of adenylate cyclase by epinephrine was unaffected. In the three subjects for whom binding measurements were made, the number of binding sites for [32P]2-MeSADP was reduced from 534 +/- 44 molecules per platelet during control and placebo periods (11 determinations) to 199 +/- 78 molecules per platelet during drug treatment (three determinations). There was no consistent change in the binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
Low-Km cyclic AMP phosphodiesterase from human platelets. Stimulation of activity by phosphorylation of the enzyme and affinity labeling of the active site.
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3',5'-Cyclic-AMP Phosphodiesterases↗
S-(4-Bromo-2,3-dioxobutyl)glutathione: a new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase.
S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 microM) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The kobs exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 microM, with a kmax of 0.078 min-1 and K1 = 66 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 1.3 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.48 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH4, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified: Lys82-His-Asn-Leu-X-Gly-Glu-Thr-Glu-Glu-Glu-Arg93, in which X is modified Cys86, and Leu109-Gln-Leu-Ala-Met-CmCys-Y-Ser-Pro-Asp-Phe-Glu-Arg121 , in which Y is modified Tyr115. Only the Lys82-Arg93 peptide was modified in the presence of S-hexylglutathione when the enzyme retained full activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide.
Pig heart NADP-specific isocitrate dehydrogenase is inactivated by N-ethylmaleimide (NEM) (Colman, R. F., and Chu, R. (1970) J. Biol. Chem. 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+. We have now treated the enzyme with [3H]NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides. In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CATITPDEAR and EPIICK, were labeled at Cys. Upon reaction with [3H]NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis. The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM. The Cys in SSGGFVWACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate. Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with [14C]iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced. Six corresponding peptides were isolated from enzyme treated under denaturing conditions with [3H]NEM. These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme. A comparison of the sequences of the NH2-terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.
Amino Acid Sequence↗
Identification of cysteine-319 as the target amino acid of 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate in bovine liver glutamate dehydrogenase.
The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) has been shown to react with bovine liver glutamate dehydrogenase in the region of the GTP-dependent NADH inhibitory site with incorporation of about 1 mol of reagent/mol of subunit [Ozturk, D. H., Safer, D., & Colman, R. F. (1990) Biochemistry 29, 7112-7118]. The modified enzyme was shown to contain only 5 free sulfhydryl groups upon 5,5'-dithiobis (2-nitrobenzoate) titration as compared with 6 in the unmodified enzyme. In the unmodified enzyme digested with trypsin, 6 cysteinyl peptides were detected by high-performance liquid chromatography upon treatment with iodo [3H]acetic acid. In contrast, only 5 (carboxymethyl)cysteinyl peptides were detected in 8-BDB-TA-5'-TP-modified enzyme. When carboxymethylated modified and unmodified enzymes were digested with thermolysin, 6 peptide sequences containing (carboxymethyl)cysteine were obtained in the unmodified enzyme, but only 5 were observed in the modified enzyme. The (carboxymethyl)cysteine which was absent in the modified enzyme was determined to be Cys-319, leading to the conclusion that 8-BDB-TA-5'-TP reacts with Cys-319, thereby preventing it from subsequent reaction with radioactive iodoacetate. It was previously reported that 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TA-5'-DP) modifies Cys-319 in this enzyme [Batra, S. P., & Colman, R. F. (1986) Biochemistry 25, 3508-3515].(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Evaluation of cysteine 283 and glutamic acid 284 in the coenzyme binding site of Salmonella typhimurium glutamate dehydrogenase by site-directed mutagenesis and reaction with the nucleotide analogue 2-[4-bromo-2,3-dioxobutyl)thio)-1,N6-ethenoadenosine 2',5'-bisphosphate.
NADP(+)-specific glutamate dehydrogenase of Salmonella typhimurium was previously shown to react irreversibly at the coenzyme site with the nucleotide analogue 2-((4-bromo-2,3-dioxobutyl)thio)-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A 2',5'-DP) yielding a partially active enzyme, and inactivation was attributed to modification of the peptide Leu282-Cys-Glu-Ile-Lys286 (Bansal, A., Dayton, M.A., Zalkin, H., and Colman, R.F. (1989) J. Biol. Chem. 264, 9827-9835). Three mutant enzymes have now been engineered, expressed in Escherichia coli, and purified: the single mutants C283I and E284Q and the double mutant C283I:E284Q. The wild-type and mutant enzymes have similar specific activities and Km values for alpha-ketoglutarate, ammonium ion, and NADPH, indicating that neither cysteine 283 nor glutamic acid 284 is essential for activity. The mutant enzyme E284Q, like wild-type glutamate dehydrogenase, is substantially inactivated by 2-BDB-T epsilon A 2',5'-DP. In contrast, the two cysteine mutant enzymes, C283I and C283I:E284Q, are not inactivated by 2-BDB-T epsilon A 2',5'-DP. Modified tryptic peptides with the sequence Leu-X-Glu(Gln)-Ile-Lys were isolated from wild-type or E284Q enzymes inactivated by 2-BDB-T epsilon A 2',5'-DP. This peptide was absent from digests of active wild-type enzyme modified in the presence of the protectant NADPH and from digests of active C283I enzyme after incubation with 2-BDB-T epsilon A 2',5'-DP. Although it is not required for catalytic activity, cysteine 283 is implicated by the results of the affinity labeling experiments as the reaction target of the nucleotide analogue and is located in the region of the coenzyme binding site.
Adenosine Diphosphate↗
High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.
In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.
Adenosine Triphosphate↗
Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase.
Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of alpha 2 beta gamma, with the alpha subunit exhibiting a molecular weight of 39,000 and the beta and gamma each having molecular weights of 41,000. The amino-terminal sequences (33-35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the alpha subunit by 11-12 amino acids relative to that of the larger beta and gamma subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40,000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the alpha subunit. The beta and gamma subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the beta and gamma subunits. The three subunits appear to be closely related, but beta and gamma are more similar to each other than either is to the alpha subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41-69]. It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the alpha beta and alpha gamma dimers are nonidentical but functionally similar.
Amino Acid Sequence↗
Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate.
Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.
Adenosine Diphosphate↗
Conformations of the coenzymes and the allosteric activator, ADP, bound to NAD(+)-dependent isocitrate dehydrogenase from pig heart.
NAD(+)-dependent isocitrate dehydrogenase from pig heart is an allosteric enzyme that is activated by ADP and is inhibited by NADPH in the presence of NADH. Transferred nuclear Overhauser effect measurements, made at a range of times to ensure that observed effects are due to direct dipole-dipole transfer and not to spin diffusion, were used to determine the conformations of pyridine nucleotide coenzymes and of the allosteric effector ADP. For NAD+, significant effects were observed on the N2 proton (on the nicotinamide ring) when the N1' proton (on the nicotinamide ribose) was saturated and on the N6 proton when the N2' proton was saturated, indicating that the conformation of the nicotinamide-ribose moiety is anti. The anti conformation is expected because of the stereospecificity of NAD(+)-dependent isocitrate dehydrogenase and is the same as for NADP(+)-dependent isocitrate dehydrogenase. For the adenosine moiety of NAD+, the predominant nuclear Overhauser effect on the A8 proton is found when the A2' proton is saturated. This result implies that the adenine-ribose bond is anti with respect to the ribose. Previous kinetic and binding studies of ADP activation have shown an influence of divalent metal ions. The conformation of bound ADP, in the presence of Mg2+ and/or Ca2+, is found to be anti about the adenine-ribose bond. The 3'H-8H distance increases when Ca2+ is added to the Mg-ADP-enzyme complex. Changes in the 4'H-1'H distance upon addition of isocitrate are indicative of interactions between the ADP activator site and the isocitrate site.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate↗
Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate.
The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K+, and Mn2+ and only 1 mol of reagent/mol of subunit is incorporated [DeCamp, D.L., Lim, S., & Colman, R.F. (1988) Biochemistry 27, 7651-7658]. We have now identified the resultant modified residues. After reaction with 8-BDB-TA-5'-TP at pH 7.0, modified enzyme was incubated with [3H]NaBH4 to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5'-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn162-Ile-Cys-Lys165 and Cys151-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys161, with a smaller amount of Asn43-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg55. Reaction in the presence of the protectants phosphoenolpyruvate, K+, and Mn2+ yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys151 and Cys48.
Adenosine Diphosphate↗
Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer.
Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH [( S]NADPH). For native enzyme, ultrafiltration studies revealed that [S]NADPH reversibly occupies about two sites/enzyme subunit in the absence of other ligands; by the addition of ADP, [S]NADPH binding can be limited to one molecule/subunit. The enzyme is irreversibly inactivated by reaction with 4-(iodoacetamido)salicylic acid (ISA) at lysine126 within the 2-oxoglutarate binding site [Holbrook, J.J., Roberts, P.A. & Wallis, R.B. (1973) Biochem. J. 133, 165-171]. ISA-modified enzyme binds 1 molecule [S]NADPH/subunit in the absence of ADP, suggesting that reaction at the substrate site blocks binding at the catalytic, but not at the regulatory site. The fluorescence spectrum of ISA-modified enzyme overlaps the absorption spectrum of [S]NADPH allowing a distance measurement between these sites by resonance energy transfer. [S]NADPH quenches the emission of ISA-modified enzyme, yielding 3.2 nm as the average distance between sites. ADP competes for the [S]NADPH site but does not affect the fluorescence of ISA-modified enzyme, indicating that [S]NADPH quenching is attributable to energy transfer rather than to a conformational change. The 3.2 nm thus represents the distance between the 2-oxoglutarate and reduced coenzyme regulatory sites of glutamate dehydrogenase.
Adenosine Diphosphate↗
Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate.
Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)
3',5'-Cyclic-AMP Phosphodiesterases↗
Affinity labeling of nucleotide binding sites of enzymes and platelets.
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3',5'-Cyclic-AMP Phosphodiesterases↗
Affinity labeling of adenine nucleotide sites in enzymes.
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Adenine Nucleotides↗
Plasmin-induced platelet aggregation is accompanied by cleavage of aggregin and indirectly mediated by calpain.
We recently reported that thrombin-induced platelet aggregation 1) is accompanied by cleavage of aggregin, a 100-kDa membrane protein and a putative ADP receptor, 2) is indirectly mediated by intracellularly activated calpain, and 3) requires the occupancy of high-affinity thrombin receptors. Because of the similarities between responses after platelet activation induced by thrombin and plasmin (greater than or equal to 1.0 casein unit/ml), we investigated whether or not plasmin-induced platelet aggregation proceeds by the same mechanism that underlies thrombin-induced platelet aggregation. We found that the rate of plasmin-induced aggregation of washed intact platelets and that of platelets modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA, an affinity analogue of ADP, which covalently modifies aggregin) were similar, indicating that the aggregation is independent of the ADP effect. Plasmin completely cleaved [3H]FSBA-labeled aggregin in intact platelets. A mixture of metabolic inhibitors (2-deoxy-D-glucose, gluconolactone, and antimycin A) completely inhibited plasmin-induced platelet aggregation and plasmin-induced cleavage of aggregin, demonstrating that an energy-requiring step is involved in the reaction. The synthetic hexapeptide affinity reagent Phe-Gln-Val-Val-Cys(NpyS)-Gly-NH2 (NpyS = 3-nitro-2-thiopyridine), a potent and specific inhibitor of thrombin-induced platelet aggregation and platelet calpain, completely inhibited plasmin-induced platelet aggregation and plasmin-induced cleavage of aggregin. These results suggest that, like thrombin, plasmin-induced platelet aggregation is accompanied by the cleavage of aggregin and these responses are indirectly mediated by the intracellularly activated calpain.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
5'-p-fluorosulfonyl)benzoyl-8-azidoadenosine: a new bifunctional affinity label for nucleotide binding sites in proteins.
A new bifunctional affinity label, 5'-p-(fluorosulfonyl)benzoyl-8-azidoadenosine (5'-FSBAzA), has been synthesized by condensation of p-(fluorosulfonyl)benzoyl chloride with 8-azidoadenosine. 5'-FSBAzA has been characterized by elemental analysis, thin-layer chromatography, and ultraviolet and 1H NMR spectroscopy. The affinity label contains both an electrophilic fluorosulfonyl moiety and a photoactivatable azido group which are capable of reacting with several classes of amino acids found in enzymes. 5'-FSBAzA reacts with bovine liver glutamate dehydrogenase in a two-step process: a dark reaction yielding about 0.5 mol of the sulfonylbenzoyl-8-azidoadenosine (SBAzA) group bound/mol enzyme subunit by reaction of the enzyme at the fluorosulfonyl group, followed by photolysis in which 25% of the covalently bound SBAzA becomes crosslinked to the enzyme. 5'-FSBAzA-modified glutamate dehydrogenase, both before and after photolysis, retains full catalytic activity but is less sensitive to allosteric inhibition by GTP, to activation by ADP, and to inhibition by 1 mM NADH. These results suggest the modification in the dark reaction of a regulatory nucleotide binding site. Photoactivation of the covalently bound reagent may have general applicability in relating modified amino acids which are close to each other in the region of the purine nucleotide binding sites of glutamate dehydrogenase and other proteins.
Adenosine↗