Interaction of L- and D-3-amino-1-chloro-2-pentanone with gamma-glutamylcysteine synthetase

The optical isomers of 3-amino-1-chloro-2-pentanone, which are the alpha-chloroketone analogs of L- and D-alpha-aminobutyrate, were synthesized and found to be highly potent irreversible inactivators of gamma-glutamylcysteine synthetase. These chloroketones are 20 to 30 times more active than L-2-amino-4-oxo-5-chlorpentanoate. L- and D-Glutamate, in the presence of Mg2+ or Mn2+, protect the enzyme against inactivation. The enzyme is almost completely inhibited by cystamine under conditions in which 0.5 mol of this compound is bound/mol of enzyme. Treatment of the enzyme with cystamne, which produces inhibition that is reversible by dithiothreitol, prevents the interaction of the new chloroketones, L-2-amino-4-oxo-5-chloropentanoate and methionine sulfoximine with the enzyme. The findings suggest that a sulfhydryl group at the active site interacts with the chloroketones and with cystamine and that the chloroketone inhibitors and cystamine bind to the enzyme as glutamine analogs. The data also suggest that a gamma-glutamyl-S-enzyme intermediate may be formed in the reaction catalyzed by this enzyme.     

Inhibition by ADP of prostaglandin induced accumulation of cyclic AMP in intact human platelets

The influence of extracellular ADP on cyclic AMP accumulation within intact human platelets was studied. ADP inhibited the increase in cyclic AMP which occurs when platelets are exposed to prostaglandin E1 or I2. The degree of inhibition varied in the range 70-95% , and was half maximal at ADP concentrations of between 0.3 and 2 microM. Other naturally occurring diphosphates, i.e. GDP, IDP and UDP, were at least 100 fold less effective than ADP, and UDP at 1mM partially reversed the effect of ADP. The effect by ADP was completely reversed by ATP, but only attenuated to a minor degree of 10 mM EDTA. Increasing concentrations of ADP caused a progressive degree of inhibition of cyclic AMP accumulation, and the kinetics of this inhibition were compatible with a simple saturable process with no cooperativity. ADP added 10 seconds after prostaglandin E1 blocked cyclic AMP accumulation within 1-2 seconds, and addition of ATP after ADP and prostaglandin I2 relieved the inhibition due to ADP within 2-3 seconds. The action of ADP was blocked by sulphydryl reagents including N-substituted maleimides, cytochalasin A, NBD chloride and p-mercuribenzene sulphonate. The data were considered to be consistent with mediation of the ADP effect through a sulphydryl-bearing specific extracellular receptor coupled to the adenylate cyclase.     

Factors influencing the response of human blood platelets to analogues of ADP which may act as partial agonists at the ADP receptor.

1. The prior addition of non-aggregating concentrations of the divalent cation ionophore, A-23187, causes human platelets to aggregate in response to a subsequent addition of the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ADP (oADP and or ADP). Previous studies [Pearce et al. (1978) Eur. J. Biochem. 88, 543--555] have shown that these derivatives act as partial agonists at the platelet ADP receptor inducing only the transition from discoid to globular morphology ('shape change'). A secretion response is also observed on addition of a low concentration of ionophore A-23187 prior to orADP. These responses are not observed if ionophore A-23187 is added prior to the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ATP (oATP and or ATP) and are markedly inhibited by prior addition of the ADP antagonist, adenosine 5'-[beta, gamma-methylene]triphosphate. 2. The aggregation response to oADP in the presence of ionophore A-23187 is reduced but not eliminated by addition of 3 mM EGTA when studies are performed in heparinised platelet-rich plasma. Additions of 3 mM EGTA in citrated platelet-rich plasma, or of 4 mM EDTA in either system completely inhibits this response. Inhibitors which are reported to elevate the intracellular concentration of adenosine 3':5'-monophosphate (cyclic AMP) or to prevent Ca2+ movement also inhibit the aggregation response to oADP which is observed in the presence of ionophore A-23187. 3. Prior addition of inhibitors of adenylate cyclase fails to cause an aggregation response to subsequent addition of oADP or orADP. Certain of these inhibitors enhance and prolong the shape change response to oADP or orADP but only at concentrations an order of magnitude in excess of those required to antagonise inhibition by agents such as prostaglandin E1, which act by increasing the concentration of cyclic AMP. 4. The concentration of prostaglandin E1, adenosine or papaverine required to inhibit shape change induced by oADP is one to two orders of magnitude lower than that required to inhibit shape change induced by ADP. 5. Prior addition of oADP decreases the lag phase in the response of human platelets to arachidonate while also increasing the concentration required to observe half-maximal response, and causing a decrease in the extent of the response. Prior addition of oATP also diminishes the extent of this response and increases the concentration of arachidonate required but has no effect on the lag phase. 6. The data suggest that oADP and orADP are capable only of acting as partial agonists at the ADP receptor because of a defective ability to increase cytosolic Ca2+ concentration. The defect is rectified by the presence of low concentrations of ionophore A-23187, which promotes mobilisation of Ca2+ from an intracellular store. The results do not appear consistent with the thesis that a decrease in platelet cyclic AMP is an initiating event in aggregation induced by ADP, but do support a model which implicates cyclic AMP in depletion of cytosolic Ca2+.     

Effect of N-2, 2'-O-dibutyryl cyclic AMP upon the interconvertible forms of cyclic AMP phosphodiesterase from human platelets.

The influence of dibutyryl cyclic AMP upon the equilibrium existing between three interconvertible forms - 2.5 S, 4.8 S and 7 S - of cyclic AMP phosphodiesterase from human platelets was investigated. It shifted the equilibrium towards the lighter form. It also exerted a protective effect against the thermo-inactivation of the enzyme. It is suggested that the analogue-induced equilibrium shift towards the dissociated high Km form of the phosphodiesterase might reflect a regulatory mechanism occurring also with natural cyclic nucleotides.     

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.     

Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase.

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.     

Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platelets

1. Human platelet-rich plasma prelabelled with [(3)H]adenine was incubated at 37 degrees C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and P(i), and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [(3)H]ATP to [(3)H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP-hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [(3)H]ADP and [(3)H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [(3)H]ATP disappeared rapidly during the combined shape change-aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.     

Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets

Prostacyclin (PGX) strikingly increases cyclic AMP concentrations in human platelets. Prostacyclin is approximately 10 times more active than PGD₂, 30 times more active than PGE₁ and more than 1000 times more active than its stable end product, 6-oxo-PGF_1α.These results correlate well with the anti-aggregating activity of prostacyclin, compared with PGE₁ and PGD₂.     

Spin-labeled analogues of ATP, ADP and AMP: substitutes for normal nucleotides in biochemical systems

The different roles and effectiveness of adenosine monophosphate, diphosphate and triphosphate labeled at the 6 position of the purine ring with 2,2,6,6-tetramethylpiperidine-1-oxyl in reactions catalyzed by Escherichia coli glutamine synthetase (GS) have been investigated. Our results show that the spin-labeled ATP (Tempo-ATP) serves as a substrate in the glutamine synthesis reaction and in the adenylation of E. coli glutamine synthetase catalyzed by ATP: glutamine adenylyl transferase (ATase) with essentially the same effectiveness as normal ATP. In another reaction (gamma-glutamyltransferase), Tempo ADP serves as an effector with a Km of 9.4 . 10(-8) M compared to 1.2 . 10(-8) M for the normal ADP, while covalently bonded Tempo-AMP serves as a modifier on the catalytic properties of E. coli glutamine synthetase just as the covalently bonded normal AMP does. The dissociation constants between the labeled nucleotides, Mn2+, Mg2+ and Ca2+ are in the same order of magnitude as the binding constants for those cations and the corresponding normal nucleotides. Our findings indicate that the spin-labeled nucleotides are good substitutes for the normal nucleotides in the biochemical systems studied.     

2-(N-acyl) and 2-N-acyl-N(6)-substituted analogues of adenosine and their affinity at the human adenosine receptors

A series of 2-(N-acyl) and 2-(N-acyl)-N(6)-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A(1), A(2A), and A(3) receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A(2A) and A(3) receptors, while the N(6)-cyclopentyl substituent in 4h and 4i induced high potency [A(1) (K(i))=20.7 and 31.8 nM respectively] at the A(1) receptor and resulted therefore in increased selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.     

Synthesis of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine analogues: succinamide, L-2-hydroxysuccinamide, and L-2-hydroxysuccinamic acid hydrazide analogues

The syntheses of three analogues of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine are described. N-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)succinamide was synthesized by the reaction of pentafluorophenyl succinamate with 2-acetamido-2-deoxy-beta-D-glucopyranosylamine. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosylamine was synthesized, and the complete assignment of the 1H NMR spectrum is given. Reaction of the protected beta-D-glycosylamine with L-malic acid chloralid in the presence of a coupling agent (EEDQ) gave N4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-L-malamic acid chloralid that was deprotected two ways: (1) using ammonia, which gave N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-2-hydroxysuccinamide, and (2) using hydrazine, which gave N4-(2-acetamido-2-deoxy-1-D-glucopyranosyl)-L-2-hydroxysuccinamic acid hydrazide.     

Both the D-(+) and L-(-) enantiomers of nicotine inhibit Abeta aggregation and cytotoxicity

The underlying cause of Alzheimer's disease is thought to be the aggregation of monomeric beta-amyloid (Abeta), through a series of toxic oligomers, which forms the mature amyloid fibrils that accumulate at the center of senile plaques. It has been reported that L-(-)-nicotine prevents Abeta aggregation and toxicity, and inhibits senile plaque formation. Previous NMR studies have suggested that this could be due to the specific binding of L-(-)-nicotine to histidine residues (His6, His13, and His14) in the peptide. Here, we have looked at the effects of both of the L-(-) and D-(+) optical enantiomers of nicotine on the aggregation and cytotoxicity of Abeta(1-40). Surprisingly, both enantiomers inhibited aggregation of the peptide and reduced the toxic effects of the peptide on cells. In NMR studies with Abeta(1-40), both enantiomers of nicotine were seen to interact with the three histidine residues. Overall, our data indicate that nicotine can delay Abeta fibril formation and maintain a population of less toxic Abeta species. This effect cannot be due to a highly specific binding interaction between nicotine and Abeta, as previously thought, but could be due instead to weaker, relatively nonspecific binding, or to the antioxidant or metal chelating properties of nicotine. D-(+)-nicotine, being biologically much less active than L-(-)-nicotine, might be a useful therapeutic agent.     

Cyclic AMP inhibits synthesis of prostaglandin endoperoxide (PGG2) in human platelets.

Dibutyryl-cAMP but not dibutyryl-cGMP inhibited platelet aggregation and release of ¹⁴C-serotonin and ADP when induced by collagen and arachidonate but not when induced by the endoperoxide PGG₂^* (TXB₂) induced by addition of collagen to platelet rich plasma (PRP) was decreased by dibutyryl-cAMP and agents known to increase the concentration of cAMP (PGE₁, PGD₂, theophylline and acetyl choline).PGE₂ in concentrations known to decrease cAMP levels increased the formation of TXB₂ whereas concentrations of PGE₂ known to increase cAMP levels decreased the amount of TXB₂ formed. That this was due to an effect on the cyclooxygenase was indicated by inhibition of the transformation of arachidonic acid by DB-cAMP and by high concentrations of PGE₂. Additional support for regulation of the cyclo-oxygenase by cAMP and its relevance to platelet aggregation was obtained by demonstrating stimulation of PGG₂ induced aggregation by low concentrations of PGE₂ and the absence of this effect in the presence of a cyclo-oxygenase inhibitor.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

Prostaglandin E2: Effects on aggregation,shape change and cyclic AMP of rat platelets

PGE₂ inhibits intracellular cyclic AMP accumulation induced by PGE₁ in rat platelets. PGE₂ also counteracts PGE₁-inhibition of both platelet aggregation and shape change. However, in the presence of theophylline, PGE₂ acts like PGE₁ in inhibiting aggregation. The mechanism of interaction of the two closely related prostaglandins is discussed.     

The effect of ADP, calcium and some inhibitors of platelet aggregation on protein phosphokinases from human blood platelets.

A protein phosphokinase (ATP: protein phosphotransferase EC 2.7.1.37) which is stimulated by 3',5'-cyclic adenosine monophosphate (cyclic AMP) has been partially purified from both the cytoplasmic and membrane fractions of human platelets. The kinetics of both enzymes preparations are similar in respect to cyclic AMP, ATP, ADP and AMP. 5-10-minus 7 M cyclic AMP stimulated both preparations by approximately 100%. Both ADP and AMP at a concentration of 5-10-minus 5 M inhibited protein phosphokinase activity of the soluble and membrane preparation by between 50% and 70%. The response of the two enzyme preparations to calcium differed. 10 mM Ca-2+ inhibited soluble protein phosphokinase activity approximately 80% both in the presence and absence of 5-10 minus 7 M cyclic AMP whereas the same concentrations of Ca-2+ inhibited the membrane-bound enzyme by approximately 60% in the presence of 5-10-minus 7 M cyclic AMP and 40% in the absence of cyclic AMP. This observation may be of importance in understanding the mechanism of platelet aggregation.     

Stimulus-secretion coupling in platelets. Effects of drugs on secretion on adenosine 5'-triphosphate.

The mechanism of stimulus-secretion coupling in platelets was investigated by observing the effects of drugs on the kinetics on ATP secretion induced by either thrombin or the divalent cation ionophore A23187. The actual secretion is the same with either of these agents, since the rate constants and activation energies of secretion are the same and since drugs that affect the final, enzyme-independent steps of thrombin-induced secretion have the same effect on ionophore-induced secretion. Drugs that affect early steps of thrombin-induced secretion have no effect on ionophore-induced secretion. Drugs that act through cAMP (PGE1, theophylline, dibutyryl-cAMP) slow an early step in the mechanism of thrombin-induced secretion and completely block at higher levels, with the required concentration of inhibitor dependent on thrombin concentration. The inhibition of rate appears to be all-or-none, with no intermediate rates observed. By replacing thrombin with trypsin, which makes it possible to observe a complete change in rate-determining step from an enzyme-dependent to an enzyme-independent platelet step, it was found that these drugs slow the rate only when the enzyme-independent step is rate determining. These drugs have no effect on A23187-induced secretion. It was concluded that cAMP inhibits at a step after the enzyme step but before the final step by interfering with transmission of the stimulus-secretion coupling signal. Disruption of microfilament function by cytochalasin B (10 muM) accelerates the rate of secretion induced by either thrombin or ionophore. The microtubule agents colchicine, vinblastine, and vincristine had effects only at concentrations above those usually considered necessary for the specific inhibition of microtubule function. Drugs that inhibit prostaglandin synthesis (aspirin, indomethacin, eicosatetraynoic acid), drugs that block ATP production (antimycin A, deoxyglucose), or several other drugs previously reported to inhibit platelet function had no effect on secretion.