Isolation and characterization of an acyl carrier protein from pigeon liver fatty acid synthetase by controlled proteolysis with elastase.

Controlled proteolytic cleavage of 4'-phospho[14C]pantetheine-labeled pigeon liver fatty acid synthetase generates two 4'-phospho[14C]pantetheine-labeled peptides, Ec1 and Ec2. These are separated from each other and the core enzyme by gel permeation chromatography on a Sephadex G-75 column. The two radioactively labeled peptides constitute 50% of the radioactivity initially present in the 4'-phospho[14C]pantetheine-labeled fatty acid synthetase. The remaining label in the core enzyme is released quantitatively by proteolytic cleavage with trypsin. The molecular weights of Ec1 and Ec2 peptides, as determined by size exclusion chromatography and SDS-polyacrylamide gel electrophoresis, are 12000 and 6000, respectively. Both the higher and lower molecular weight peptides are homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. The higher molecular weight peptide, Ec1, is characterized as an acyl carrier protein by the transacylation reaction between the unlabeled Ec1 peptide and radioactively labeled acetyl- and malonyl-CoA. Since Ec2 peptide also contains the prosthetic group present in the Ec1 peptide, the Ec2 peptide appears to result from the proteolytic cleavage of the higher molecular weight peptide, Ec1. Amino acid composition of the acyl carrier protein shows the presence of 1 mol of 4'-phosphopantetheine per mol of protein. 2 mol of acyl carrier protein are present per mol of the fatty acid synthetase. The amino acid analysis is in good agreement with the molecular weight of the Ec1 peptide, as determined by gel filtration and SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid analysis of this peptide shows the presence of an arginine residue.

Inactivation of yeast hexokinase by o-phthalaldehyde: evidence for the presence of a cysteine and a lysine at or near the active site.

Yeast hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a homodimer, was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics over a wide range of the inhibitor concentration. The second-order-rate constant for the inactivation of hexokinase was estimated to be 45 M-1.s-1. Hexokinase was protected more by sugar substrates than by nucleoside triphosphates during inactivation by o-phthalaldehyde. Absorption spectrum (lambda max 338 nm), and fluorescence excitation (lambda max 363 nm) and emission (lambda max 403 nm) spectra of the hexokinase-o-phthalaldehyde adduct were consistent with the formation of an isoindole derivative. These results also suggest that sulfhydryl and epsilon-amino functions of the cysteine and lysine residues, respectively, participating in the isoindole formation are about 3 A apart in the native enzyme. About 2 mol of the isoindole per mol of hexokinase dimer were formed following complete loss of the phosphotransferase activity. Chemical modification of hexokinase by iodoacetamide in the presence of mannose resulted in the modification of six sulfhydryl groups per mol of hexokinase with retention of the phosphotransferase activity. Subsequent reaction of the iodoacetamide modified hexokinase with o-phthalaldehyde resulted in complete loss of the phosphotransferase activity with concomitant modification of the remaining two sulfhydryl groups of hexokinase. Chemical modification of hexokinase by iodoacetamide in the absence of mannose resulted in complete inactivation of the enzyme. The iodoacetamide inactivated hexokinase failed to react with o-phthalaldehyde as evidenced by the absence of a fluorescence emission maximum characteristic of the isoindole derivative. The holoenzyme failed to react with [5'-(p-fluorosulfonyl)benzoyl]adenosine. The dissociated hexokinase could be inactivated by [5'-(p-fluorosulfonyl)benzoyl]adenosine; the degree of inactivation paralleled the extent of reaction between o-phthalaldehyde and the nucleotide-analog modified enzyme. Thus, it is concluded that two cysteines and lysines at or near the active site of the hexokinase were involved in reaction with o-phthalaldehyde following complete loss of the phosphotransferase activity. An important finding of this investigation is that the lysines, involved in isoindole formation, located at or near the active site are probably buried.(ABSTRACT TRUNCATED AT 400 WORDS)

Liberation, purification, and properties of thioesterase component of pigeon liver fatty acid synthetase complex.

Proteolysis of pigeon liver fatty acid synthetase with elastase results in the quantitative cleavage of the thioesterase component from the enzyme complex. This thioesterase component is two or three times more active catalytically in the isolated state than in the native fatty acid synthetase, and its activity is not affected by the presence or absence of reducing thiols. The proteolytically cleaved thioesterase is separated from the core enzyme in one step by size-exclusion chromatography on a Sephadex G-75 column. The peptide obtained by gel permeation is homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. Size-exclusion chromatography on Bio-Gel A 0.5 m and Sephadex G-75 columns, sucrose density gradient ultracentrifugation, and N-terminal amino acid analysis also indicate that the proteolytically cleaved thioesterase is homogeneous. The sedimentation coefficient of the thioesterase is approximately 2.9 S. Proteolytic cleavage with elastase also quantitatively releases the [1,3-14C]- or [1,3-3H]diisopropylphosphofluoridate-labeled thioesterase component from the correspondingly labeled fatty acid synthetase. Binding studies with 14C- or 3H-labelled diisopropylphosphofluoridate and fatty acid synthetase show that 2 mol of the label are bound per mol of the enzyme when complete loss of fatty acid-synthesizing activity occurs. The molecular weight of the thioesterase component is estimated to be 36000 by size-exclusion chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis.

Phospholipase A2: its role in ADP- and thrombin-induced platelet activation mechanisms.

ADP and thrombin are two of the most important agonists of platelet aggregation--a cellular response that is critical for maintaining normal hemostasis. However, aberrant platelet aggregation induced by these agonists plays a central role in the pathogenesis of cardiovascular and cerebrovascular diseases. Agonist-induced primary or secondary activation of phospholipases leads to generation of the second messengers that participate in biochemical reactions essential to a number of platelet responses elicited by ADP and thrombin. Phospholipase A2 (PLA2) has been linked to cardiovascular diseases. However, the mechanism(s) of activation of PLA2 in platelets stimulated by ADP and thrombin has remained less well defined and much less appreciated. The purpose of this review is to examine and compare the molecular mechanisms of activation of PLA2 in platelets stimulated by ADP and thrombin.

Proton magnetic resonance spectroscopic analysis of diadenosine 5',5"'-polyphosphates.

Certain diadenosine 5',5"'-polyphosphates are potent inhibitors of ADP stimulated platelet aggregation, acting possibly via competitive ADP-receptor binding. 1H NMR studies of a series of such compounds where the number of phosphate groups between adenosine groups was varied from 2 to 6 were performed to analyze possible preferred solution conformations and to define structure-activity relations. Relative to mononucleotides ADP and ATP, chemical shifts of adenosine proton resonances in diadenosine polyphosphate analogs are upfield shifted suggesting base stacking. This effect is greatest for AP2A and AP3A. Coupling constants of ribose ring proton resonances support the idea of an anti-base-ribose ring conformation, and 3JH5'-P values suggest a preferred gauche H-C-O-P structure. In all cases, NMR parameters for AP2A are near-limiting values for a static base stacked conformation. Increasing the number of phosphate groups between adenosine moieties tends to weaken this interaction.

ADP-induced platelet aggregation and inhibition of adenylyl cyclase activity stimulated by prostaglandins: signal transduction mechanisms.

ADP is the oldest and one of the most important agonists of platelet activation. ADP induces platelet shape change, exposure of fibrinogen binding sites, aggregation, and influx and intracellular mobilization of Ca2+. ADP-induced platelet aggregation is important for maintaining normal hemostasis, but aberrant platelet aggregation manifests itself pathophysiologically in myocardial ischemia, stroke, and atherosclerosis. Another important aspect of ADP-induced platelet activation is the ability of ADP to antagonize adenylyl cyclase activated by prostaglandins. ADP-induced inhibition of the stimulated adenylyl cyclase activity does not appear to play a role in ADP-induced platelet aggregation in vitro or in vivo. It is believed that a single ADP receptor mediates the above two ADP-induced platelet responses in platelets. The ADP receptor mediating ADP-induced platelet aggregation and inhibition of the stimulated adenylyl cyclase activity has not been purified. Therefore, the nature of molecular mechanisms underlying the two seemingly unrelated ADP-induced platelet responses remains either unclear or less well understood. The purpose of this commentary is to examine and make suggestions concerning the role of phospholipases and G-proteins in the molecular mechanisms of signal transduction underlying the two ADP-induced platelet responses. It is hoped that such discussion would stimulate thinking and invite future debates on this subject, and energize investigators in their efforts to advance our knowledge of the details of the molecular mechanisms of ADP-induced platelet activation.

Specificity of the sequence in Phe-Gln-Val-Val-Cys (-3-nitro-2-pyridinesulfenyl)-Gly-NH2--a selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation is mediated in part by the intracellularly activated calpain expressed onto the external side of the membrane. We have previously shown that P1, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2 [Npys = 3-nitro-2-pyridinesulfenyl], an affinity analog corresponding to the highly conserved sequence Gln-Val-Val-Ala-Gly-NH2, present in domains 2 and 3 of human kininogens, was an irreversible inhibitor of platelet calpain (second-order rate constant = 5.85 mM-1 s-1). P1 also selectively blocked thrombin-induced platelet aggregation. We have now synthesized twenty-three other peptides, analogous to P1, and evaluated them to define the specificity of the amino acid sequence in P1 to selectively block thrombin-induced platelet aggregation. We find that replacement by Leu of Val and by Tyr of Phe adjacent to Gln is minimally tolerated and the resulting peptides are partially effective in selectively blocking thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. Extensions of the N-terminal sequence in P1 did not improve its selectivity. Ac-Ala-Gln-Val-Val-Ala-Gly-NH2 (Ac, acetyl), a peptide containing the conserved sequence but lacking the Npys function, neither inhibited platelet calpain nor platelet aggregation induced by thrombin. Presence of the peptide sequence and Npys function are both required in P1 for its selective action in inhibiting platelet aggregation induced by thrombin.

Aggregation of washed platelets by plasminogen and plasminogen activators is mediated by plasmin and is inhibited by a synthetic peptide disulfide.

Plasmin is known to activate platelets. However, it is not clear whether plasminogen activators as used in thrombolytic therapy can aggregate platelets and how this relates to the ability of each activator to convert plasminogen to plasmin. Urokinase (UK) and streptokinase (SK) activated purified plasminogen (2 microM) in a concentration-dependent manner. The rates of aggregation of washed platelets by the above plasminogen activators and plasminogen were similar to the extent of activation of plasminogen to plasmin in the absence of platelets. UK or SK (0.2 microM) and plasminogen (2 microM) aggregated platelets modified by an ADP affinity analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), and cleaved aggregin, a putative ADP receptor, in [3H]FSBA-modified platelets. These results suggest that the effect was independent of ADP. In contrast, incubation mixtures containing only plasminogen (2 microM) and single chain tissue plasminogen activator (sc-tPA) (less than or equal to 0.12 microM) neither activated the zymogen to an appreciable extent nor aggregated platelets. But, in the presence of fibrin(ogen) fragments (tPA-stimulator), a mixture of plasminogen and sc-tPA aggregated unmodified and FSBA-modified platelets, and cleaved aggregin. The results imply that platelets, in the presence of t-PA stimulator, potentiate activation of plasminogen to plasmin by t-PA, as previously reported. P1, Phe-Gln-Val-Val-Cys-(NpyS)-Gly-NH2, (NpyS = 3-nitro-2-thiopyridine), a synthetic hexapeptide capable of binding to and inhibiting calpain, has been shown to inhibit platelet aggregation induced by purified plasmin. P1 inhibited platelet aggregation by plasminogen and any of the three plasminogen activators. Our results show that at plasma concentrations of plasminogen and at levels of UK and SK attained after infusion of these agents during thrombolysis, these mixtures can cause maximum aggregation which may contribute to reocclusion and stenosis following infarct therapy. P1 can effectively inhibit platelet aggregation under such conditions.

Reocclusion after thrombolytic therapy: strategies for inhibiting thrombin-induced platelet aggregation.

The use of first generation plasminogen activators, urokinase, streptokinase and tissue plasminogen activator has revolutionized thrombolytic therapy for myocardial infarction and ischaemia, and potentially stroke. However, thrombolytic therapy employing these activators is limited by reocclusion of the very arteries being opened, which follows in a small but significant number of patients. The development of second generation plasminogen activators, e.g. staphylokinase and anisoylated plasminogen streptokinase activator complex, has not alleviated the problems encountered with classical plasminogen activators. It is now widely recognized that aberrant platelet aggregation induced primarily by thrombin, rather than plasmin, is one of the major causes of recurrent thrombosis following pharmacologic thrombolysis. Agents that (a) inhibit enzymatic and/or coagulant activity of thrombin, (b) block binding of thrombin to its receptor, and (c) interfere with the generation of thrombin by the prothrombinase complex may compromise haemostasis resulting in haemorrhage. We recently demonstrated that thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a putative ADP-receptor on the platelet surface, and that these events are indirectly mediated by intracellularly activated calpain expressed on the surface. In this review, we discuss the known mechanisms of thrombin-induced platelet aggregation and suggest relative advantages of potential pharmacological agents, being developed in our laboratory, over those that have been previously developed and tested. These inhibitors selectively prevent aggregation of platelets induced by thrombin by inhibiting calpain expressed on the surface. Moreover, one of these inhibitors which blocks thrombin-induced platelet aggregation does not interfere with other platelet responses mediated by thrombin or platelet aggregation induced by other agonists, such as, ADP, collagen, phorbol myristate acetate and thromboxane A2 mimetics. This selectivity could reduce the chances of perturbing the formation of a haemostatic plug.

Reaction of low molecular weight aminothiols with o-phthalaldehyde.

o-Phthalaldehyde has been recently shown to be a useful reagent for chemical modification of cyclic nucleotide dependent protein kinases, hexokinase, and fructose-1,6-bisphosphatase. It reacts covalently with closely spaced (approximately 3 A) sulfhydryl and epsilon-amino functions of cysteine and lysine residues, respectively, of these enzymes to yield fluorescent isoindole derivatives. We have found the reagent to be equally useful to investigate the degree of reactivity of sulfhydryl and amino functions in substances that do not possess enzymatic activity, e.g., glutathione, homocysteine, and cysteine. The kinetics of the reaction of nonenzymatic aminothiols with o-phthalaldehyde can be followed rapidly and conveniently by continuously monitoring the increase in relative fluorescence of the isoindole derivatives. The fluorescence emission maxima of the o-phthalaldehyde adducts can be used to compute molar transition energies that provide qualitative but useful information concerning the degree of polarity of microenvironment of the sulfhydryl and amino functions participating in isoindole formation. The kinetic and spectral data obtained from the reaction between o-phthalaldehyde and nonenzymatic low molecular weight aminothiols may be helpful in comparing the reactivities of the sulfhydryl and amino functions in enzymes.

ADP-induced platelet activation.

Platelet activation is central to the pathogenesis of hemostasis and arterial thrombosis. Platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is the first known and an important agonist for platelet aggregation. ADP not only causes primary aggregation of platelets but is also responsible for the secondary aggregation induced by ADP and other agonists. ADP also induces platelet shape change, secretion from storage granules, influx and intracellular mobilization of Ca2+, and inhibition of stimulated adenylyl cyclase activity. The ADP-receptor protein mediating ADP-induced platelet responses has neither been purified nor cloned. Therefore, signal transduction mechanisms underlying ADP-induced platelet responses either remain uncertain or less well understood. Recent contributions from chemists, biochemists, cell biologists, pharmacologists, molecular biologists, and clinical investigators have added considerably to and enhanced our knowledge of ADP-induced platelet responses. Although considerable efforts have been directed toward identifying and cloning the ADP-receptor, these have not been completely successful or without controversy. Considerable progress has been made toward understanding the mechanisms of ADP-induced platelet responses but disagreements persist. New drugs that do not mimic ADP have been found to inhibit fairly selectively ADP-induced platelet activation ex vivo. Drugs that mimic ADP and selectively act at the platelet ADP-receptor have been designed, synthesized, and evaluated for their therapeutic efficacy to block selectively ADP-induced platelet responses. This review examines in detail the developments that have taken place to identify the ADP-receptor protein and to better understand mechanisms underlying ADP-induced platelet responses to develop strategies for designing innovative drugs that block ADP-induced platelet responses by acting selectively at the ADP-receptor and/or by selectively interfering with components of ADP-induced platelet activation mechanisms.

Immunoaffinity method to identify aggregin, a putative ADP-receptor in human blood platelets.

The ADP-receptor on the surface of human platelets and cells of megakaryocytic lineage has been classified as P2T purinergic receptor for which ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has neither been purified nor cloned. We have developed an immunoaffinity method for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal antibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutamate dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled platelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated with ADP and ATP or covalently modified by the chemically reactive ADP-affinity analogs, 2- and 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (2- and 8BDB-TADP) and 2-(3-bromo-2-oxopropylthio)adenosine-5'-diphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibit ADP-induced platelet responses by selectively and covalently modifying aggregin (100 kDa), an ADP-receptor in intact human blood platelets. The results show that polyclonal antibody to FSBA-labeled GDH is capable of recognizing FSBA-labeled aggregin on platelets and, thus, could be used to purify aggregin by immunoaffinity column chromatography. The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses.

Thrombin- and cathepsin G-induced platelet aggregation: effect of protein kinase C inhibitors.

Two serine proteases, thrombin and cathepsin G, are potent agonists of human platelet activation. These pathophysiological proteases induce similar platelet responses, e.g., aggregation, shape change, and secretion of the dense granules. Maintenance of proteolytic function and the ability to bind to receptors on the platelet surface membrane are required for the responses elicited by both proteases. Protein kinase C (PKC) is a signal-transducing enzyme that is an important regulator of postreceptor intracellular changes following exposure of platelets to thrombin and cathepsin G. Inhibitors of purified PKC, e.g., staurosporine and calphostin C, have been frequently used to elucidate biochemical mechanisms mediated by the intracellular PKC following platelet activation by proteases. However, the effect of the PKC inhibitors on the amidolytic activity and on the ability of the two bioregulatory proteases to bind to cells has never been investigated. We found that staurosporine (1 and 1.5 microM), calphostin C (10 and 60 microM), and fisetin (200 and 220 microM), the three most commonly used and biochemically well-characterized inhibitors of purified PKC, completely inhibited thrombin-induced (2 nM) and cathepsin G-induced (0.85 microM) aggregation of washed human platelets, respectively. Each of the three PKC inhibitors completely blocked platelet shape change induced by thrombin (1 nM). Only fisetin inhibited platelet shape change induced by cathepsin G (0.5 microM). Only fisetin partially inhibited amidolytic activity of thrombin. The three PKC inhibitors had no inhibitory effect on the amidolytic activity of those concentrations of cathepsin G that cause maximum platelet aggregation and platelet shape change. The three PKC inhibitors completely blocked binding of 125I-thrombin to washed platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of fructose-1,6-bisphosphatase by o-phthalaldehyde.

Rabbit liver fructose-1,6-bisphosphatase, a tetramer of identical subunits was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The second-order rate constant for the inactivation was 30 M-1s-1. Fructose-1,6-bisphosphatase was completely protected from inactivation by the substrate--fructose-1,6-diphosphate but not by the allosteric effector--adenosine monophosphate. The absorption spectrum (lambda max 337 nm) and, fluorescence excitation (lambda max 360 nm) and fluorescence emission spectra (lambda max 405 nm) were consistent with the formation of an isoindole derivative in the subunit between a cysteine and a lysine residue about 3A apart. About 4 isoindole groups per mol of the bisphosphatase were formed following complete loss of the phosphatase activity. This suggests that the amino acid residues of the biphosphatase participating in reaction with o-phthalaldehyde more likely reside at or near the active site instead of allosteric site. The molar transition energy of fructose-1,6-bisphosphatase--o-phthalaldehyde adduct was estimated 121 kJ/mol and compares favorably with 127 kJ/mol for the synthetic isoindole, 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl) isoindole in hexane. It is, thus, concluded that the cysteine and lysine residues participating in isoindole formation in reaction between fructose-1,6-bisphosphatase and o-phthalaldehyde are located in a hydrophobic environment.

Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a 'half-of-the-sites' mechanism.

Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of ADP-induced platelet activation by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole: covalent modification of aggregin, a putative ADP receptor.

ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-CI) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-CI. NBD-CI inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-CI also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-CI did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-CI blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I2). NBD-CI was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [14C]-NBD-CI showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [14C]-NBD-CI. These results (1) indicate that covalent modification of aggregin by NBD-CI contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin.

Inactivation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations.

Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by 'Kitz-Wilson'-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation, purification, and characterization of a peptide that contains the beta-ketoacyl reductase, enoyl reductase, and beta-hydroxyacyl dehydrase activities of the pigeon liver fatty acid synthetase.

Controlled proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides that are designated II and IV. After 5 h of proteolysis the incubation mixture containing these peptides retains all of the component enzyme activities of the fatty acid synthetase complex. The two peptides are then separated by chromatography on an Affi-Gel Blue column. Gel filtration of the fraction containing peptide II yields a homogeneous peptide as shown by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of this peptide has been estimated to be 130 000 by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, size exclusion chromatography, and amino acid analysis. The sedimentation coefficient for peptide II is approximately 7.4S. Peptide II contains the domains for the beta-ketoacyl and enoyl reductases and beta-hydroxyacyl dehydrase activities of the fatty acid synthetase complex.