Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene.

Protein tyrosine phosphatase-1B (PTP-1B) has been implicated in the negative regulation of insulin signaling. Disruption of the mouse homolog of the gene encoding PTP-1B yielded healthy mice that, in the fed state, had blood glucose concentrations that were slightly lower and concentrations of circulating insulin that were one-half those of their PTP-1B+/+ littermates. The enhanced insulin sensitivity of the PTP-1B-/- mice was also evident in glucose and insulin tolerance tests. The PTP-1B-/- mice showed increased phosphorylation of the insulin receptor in liver and muscle tissue after insulin injection in comparison to PTP-1B+/+ mice. On a high-fat diet, the PTP-1B-/- and PTP-1B+/- mice were resistant to weight gain and remained insulin sensitive, whereas the PTP-1B+/+ mice rapidly gained weight and became insulin resistant. These results demonstrate that PTP-1B has a major role in modulating both insulin sensitivity and fuel metabolism, thereby establishing it as a potential therapeutic target in the treatment of type 2 diabetes and obesity.

Regulation of enzyme activity by cyclic phosphorylation-dephosphorylation cascades. Thermodynamic constraints.

The currently accepted model describing regulation of enzyme activity by cyclic phosphorylation-dephosphorylation cascades (Stadtman, E.R. and Chock, P.B. (1978) Curr. Top. Cell. Reg. 13, 53-95) does not take into account the fact that the individual phosphorylation and dephosphorylation reactions are reversible. A modification of the model is described which does take into account the reversible nature of the individual modification and demodification reactions. This model predicts that, if the dephosphorylating enzyme is inactive, the phosphorylation reaction will go to equilibrium, rather than to completion, as predicted by the original model. The new model also makes it possible to take into account factors which change the [ATP]/[ADP] ratio and the equilibrium constant for hydrolysis of the phosphorylated enzyme. These factors define a variable 'window' of thermodynamically allowed values of the ratio of phosphorylated to nonphosphorylated enzyme, which is imposed upon the wider range of kinetically allowed values predicted by the original model.

Kinetic mechanism of glutathione conjugation to leukotriene A4 by leukotriene C4 synthase.

The kinetic mechanism for human leukotriene (LT) C4 synthase, a membrane-bound glutathione S-transferase, which catalyzes the conjugation of glutathione (GSH) to 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (LTA4), to form 5(S)-hydroxy-6(R)-S-glutathionyl-7,9,trans-11, 14-cis-eicosatetraenoic acid (LTC4) was investigated by initial rate kinetic studies in which concentrations of both substrates and the reversible dead-end inhibitor, 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)- methoxy]- 4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid (L-699,333) were varied. Analysis of the initial velocities of LTC4 formation in the absence of the inhibitor using non-linear regression fits of various models to the data favoured a random, rapid equilibrium mechanism, with strong substrate inhibition by LTA4, over both a compulsory ordered mechanism and a ping-pong mechanism. The estimated parameters were calculated to be Vmax = 14 +/- 4 microM/min, KLTA4 = 40 +/- 18 microM, KGSH = 0.4 +/- 0.2 mM, and a KiLTA4 = 2.3 +/- 1.7 microM for the rapid equilibrium random model. Inhibition of enzymatic activity by L-699,333 was found to be reversible as assessed by the ability of the enzyme to restore its activity by 95% upon dilution. L-699,333 was found to be a competitive inhibitor against GSH and non-competitive against LTA4. Non-linear least squares regression analysis yielded estimated parameters of Km = 0.7 +/- 0.1 mM, Vmax = 2.5 +/- 0.1 microM/min, and Ki = 0.7 +/- 0.1 microM for GSH at a fixed LTA4 concentration of 20 microM, and Km = 45 +/- 3 microM, Vmax = 4.9 +/- 0.2 microM/min, and a Ki = 5.8+/-0.4 microM for LTA4 at a fixed GSH concentration of 2 mM. The rate equation for the random equilibrium mechanism accommodates the inhibition patterns observed for L-699,333 against both substrates as revealed by kinetic fits of the inhibition data to the overall rate equation.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor.

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 26 +/- 6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 1-3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM as compared to 20 +/- 1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

Inhibition of human leukocyte 5-lipoxygenase by a 4-hydroxybenzofuran, L-656,224. Evidence for enzyme reduction and inhibitor degradation.

Detailed studies of the interaction of L-656,224 (2-[(4'-methoxyphenyl)methyl]-3-methyl-4-hydroxy-5-propyl-7- chlorobenzofuran) with 5-lipoxygenase were conducted using the enzymes from human and pig leukocytes. L-656,224 was a potent inhibitor of these 5-lipoxygenases although its efficiency varied with enzyme concentration. L-656,224 also stimulated the pseudoperoxidase activity of 5-lipoxygenase as measured by the consumption of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD), indicating that this compound can reduce the enzyme. Furthermore the inhibitor was degraded rapidly by both cell-free leukocyte extracts and purified 5-lipoxygenase after incubation with 13-HPOD, ATP and calcium ions. The degradation of L-656,224 was also observed during inhibition of the lipoxygenase reaction and occurred mainly after the initial lag phase of the reaction when hydroperoxides begin to accumulate. A single major radioactive product was formed after incubation of [3H]L-656,224 with purified 5-lipoxygenase in the presence of 13-HPOD. This product was unstable and could not be isolated. During the course of the pseudoperoxidase reaction, [3H]L-656,224 covalently labelled the enzyme, suggesting that a chemically reactive species had been formed. These data are consistent with the hypothesis that L-656,224 reduces the oxidized form of the 5-lipoxygenase to an inactive form, with degradation of the inhibitor and regeneration of the active enzyme with hydroperoxides.

Bis(N,N-dimethylhydroxamido)hydroxooxovanadate inhibition of protein tyrosine phosphatase activity in intact cells: comparison with vanadate.

We have shown previously that bis(N,N-dimethylhydroxamido)hydroxooxovanadate (DMHV) is an excellent reversible inhibitor of protein tyrosine phosphatase (PTP) in vitro. DMHV does not carry a charge under physiological pH conditions and is anticipated to permeate cell membranes more easily than vanadate. In the present study, the efficacy of DMHV as a PTP inhibitor in intact cells was compared with that of vanadate by measuring phosphotyrosine levels in various cells treated with these compounds. DMHV was more effective in increasing both the phosphotyrosine levels of various proteins in 3T3L1 fibroblasts and the level of insulin-receptor phosphorylation in CHO cells overexpressing the human insulin receptor. DMHV was about 10- to 20-fold more effective than vanadate in increasing glucose transport and glycogen synthesis in 3T3L1 adipocytes. DMHV, unlike vanadate, also inhibited PTP in Jurkat cells. The implications of these observations are discussed.

The distribution and half-life for retention of vanadium in the organs of normal and diabetic rats orally fed vanadium(IV) and vanadium(V).

The concentration of vanadium in organs of diabetic rats that had been fed vanadium, either as V(IV) or V(V), in their drinking water has been determined. The kidney was found to have the highest concentration, about 185 nmol/g wet tissue. This averages about three times higher than for the liver or spleen, for which concentrations were comparable. The lung, blood plasma, and blood cells tended to have the lowest accumulations of vanadium. A time-course study indicated that the half-life for elimination of vanadium from the bodies of vanadium-fed rats is about 12 d.

ADP-arsenate. Formation by submitochondrial particles under phosphorylating conditions.

Submitochondrial particles from beef heart mitochondria synthesize ADP-arsenate from ADP and arsenate when energized by succinate. The ADP-arsenate formed hydrolyzes rapidly and this is almost certainly the mechanism by which arsenate "uncouples" oxidative phosphorylation. When sufficient hexokinase is present, a substantial portion of the ADP-arsenate formed reacts with glucose to form glucose 6-arsenate and ADP. The glucose 6-arsenate thus formed hydrolyzes, at pH 7.5 and 30 degrees C, under the conditions used, with a rate constant of 5.5 X 10(-4) s-1 and is a substrate for glucose-6-phosphate dehydrogenase.

Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on systems regulated by tyrosine phosphorylation.

The interaction of vanadate with phenol and N-acetyltyrosine ethyl ester in aqueous solution has been studied by using 51V nuclear magnetic resonance spectroscopy. On the basis of these studies, it has been concluded that vanadate rapidly esterifies the hydroxyl group of the aromatic ring to yield a phenyl vanadate. For phenol, the equilibrium constant for this reaction in terms of the convention that the activity of liquid water is 1.0 is K1 = [phenyl vanadate]/[phenol][vanadate] = 0.97 +/- 0.02. This value is well over 4 orders of magnitude larger than estimates from the literature for the corresponding equilibrium constant for the esterification of phenol by phosphate. The equilibrium constant for esterification of the phenol moiety of N-acetyltyrosine ethyl ester is similar to that for esterification of phenol. The relevance of these observations to processes that are regulated by reversible phosphorylation/dephosphorylation of tyrosine residues is discussed, in particular the insulin-like effect of vanadate.

Calcium is not required for 5-lipoxygenase activity at high phosphatidyl choline vesicle concentrations.

5-Lipoxygenase (5-LO) catalyzes the formation of 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) and leukotriene A4 (LTA4) from arachidonic acid. Following a rise in intracellular calcium, 5-LO translocates to a membrane where it reacts with arachidonic acid via an 18 kD protein (FLAP). In vitro studies using a vesicle system of phosphatidylcholine (PC) and purified 5-LO were conducted under varying concentrations of PC and calcium. At high PC concentrations, 5-LO partitioned onto the vesicle containing arachidonic acid, resulting in product formation in the absence of calcium. Addition of calcium increased the initial rate of the reaction with a small increase in product accumulation. Dilution experiments in the absence of calcium at high PC concentrations indicated that binding of 5-LO to the vesicles is rapidly reversible. In the presence of calcium, this binding is much more favorable than without calcium. Stimulation of 5-LO activity by dithiothreitol (DTT) was more pronounced at high PC concentrations than at low PC concentrations. The requirement for ATP for maximal activity was independent of vesicle concentration. Inhibitors that functioned in the conditions of low PC with calcium present also inhibited under high PC without calcium. In the presence of PC and calcium and without substrate, the enzyme was unstable and was rapidly and irreversibly inactivated. In high PC without calcium, the enzyme was much more stable but it was still subject to turnover-dependent inactivation. Fluorescence energy-transfer experiments confirmed the kinetic findings that 5-LO could bind to the vesicle in the absence of calcium. These results show that in the absence of calcium, 5-LO can reversibly bind to the vesicle containing arachidonic acid and produce the same amount of product by a similar mechanism as observed with low PC and calcium. Calcium likely causes a conformational change that increases the affinity of the enzyme for the vesicle, but it is not strictly required for enzymatic activity and has no effect on the function of the catalytic site.

Dissociation of phosphate from beef heart mitochondrial F1-ATPase. Effect of adenine nucleotides.

The effect of exposure to adenine nucleotides on the dissociation of phosphate bound to beef heart mitochondrial F1-ATPase (F1) has been studied using a layered Sephadex centrifuge column method. The order of effectiveness of nucleotides in facilitating Pi dissociation from F1 was ATP greater than AMP-PNP much greater than ADP (where AMP-PNP indicates adenosine 5'-(beta,gamma-imido)triphosphate) when phosphate had bound to F1 as Pi from the medium. When ATP was present in amounts substoichiometric to F1, essentially all of the ATP present bound to F1, and the molar ratio of Pi released to ATP bound was near one. Under similar conditions AMP-PNP bound equally well to F1, but in order to effect similar amounts of Pi release it was necessary to use approximately 10 times as much AMP-PNP as ATP. The order of effectiveness of nucleotides in facilitating dissociation of phosphate which had bound to F1 as the gamma-phosphate of ATP was ATP = AMP-PNP much greater than ADP. In this case ATP and AMP-PNP were as effective in facilitating phosphate dissociation as was ATP in the case in which phosphate had bound to F1 as Pi. It is concluded that when phosphate has bound to F1 as Pi, binding of one molecule of ATP or more than one molecule of AMP-PNP are sufficient to facilitate dissociation of phosphate. When phosphate has bound to F1 as the gamma-phosphate of ATP, binding of one molecule of either ATP or AMP-PNP is sufficient to facilitate dissociation of phosphate.

Inhibition of phosphatase and sulfatase by transition-state analogues.

The inhibition constants for vanadate, chromate, molybdate, and tungstate have been determined with Escherichia coli alkaline phosphatase, potato acid phosphatase, and Helix pomatia aryl sulfatase. Vanadate was a potent inhibitor of all three enzymes. Inhibition of both phosphatases followed the order WO4(2-) greater than MoO4(2-) greater than CrO4(2-). The Ki values for potato acid phosphatase were about 3 orders of magnitude lower than those for alkaline phosphatase. Aryl sulfatase followed the reverse order of inhibition by group VI oxyanions. Phenol enhanced inhibition of alkaline phosphatase by vanadate and chromate but did not affect inhibition of acid phosphatase. Phenol enhanced inhibition of aryl sulfatase by metal oxyanions in all cases following the order H2VO4- greater than CrO4(2-) greater than MoO4(2-) greater than WO4(2-), and N-acetyltyrosine ethyl ester enhanced inhibition of aryl sulfatase by H2VO4- and CrO4(2-) more strongly than did phenol. It is apparent that the effectiveness of metal oxyanions as inhibitors of phosphatases and sulfatases can be selectively enhanced in the presence of other solutes. The relevance of these observations to the effects of transition metal oxyanions on protein phosphatases in vivo is discussed.

Inhibition of soybean lipoxygenase-1 by a diaryl-N-hydroxyurea by reduction of the ferric enzyme.

It has been proposed that catechols and other antioxidants inhibit lipoxygenase activity by reducing the active Fe3+ form of the enzyme [Kemal et al. (1987) Biochemistry 26, 7064-7072]. In this model, reductively inactivated lipoxygenase can be reactivated by reaction with the hydroperoxide product in a pseudoperoxidase reaction. The contribution of enzyme reduction in the inhibition of the activity of soybean lipoxygenase-1 by the reducing inhibitor N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)-urea (CPHU) has been evaluated quantitatively. The inhibition by CPHU of the oxygenation of linoleic acid to 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) was accompanied by an initial lag phase which could be eliminated by the presence of exogenous 13-HpODE at the initiation of the reaction. In addition, both 13-HpODE and CPHU were found to be consumed during the lipoxygenase reaction, indicating occurrence of both oxygenase and pseudoperoxidase reactions. When analyzed individually, both the oxygenase reaction at different linoleic acid and O2 concentrations and the pseudoperoxidase reaction at different 13-HpODE and CPHU concentrations were found to follow ping-pong kinetics. A rate equation for the lipoxygenase-catalyzed reaction in the presence of reducing agent was derived considering that the inhibition of the oxygenase reaction is the combined result of 13-HpODE consumption and formation of inactive Fe2+ enzyme due to occurrence of the pseudoperoxidase reaction. By comparing the experimental data with those predicted by the rate equation, it is concluded that the inactivation of the enzyme by reduction can quantitatively account for the inhibition caused by CPHU.

Synthesis and hydrolysis of ADP-arsenate by beef heart submitochondrial particles.

The kinetic parameters for inorganic phosphate and inorganic arsenate as substrates for the synthesis of ATP and ADP-arsenate, respectively, by beef heart submitochondrial particles have been determined. The Vm and Km values for arsenate and phosphate, as well as the Km values for ADP in the two reactions, are the same within experimental error of the measurements. These data are consistent with covalent bond formation not being the rate-limiting step for either ATP or ADP-arsenate synthesis. The hydrolysis of ATP and of ADP-arsenate was studied under conditions of net synthesis of ATP or ADP-arsenate. The apparent first order rate constant for ATP hydrolysis increased with submitochondrial particle concentration, indicating that hydrolysis of ATP was catalyzed by the submitochondrial particle preparation. Nonenzymic hydrolysis of ATP was negligible compared to enzymic hydrolysis. The apparent first order rate constant for ADP-arsenate hydrolysis did not vary with submitochondrial particle concentration, indicating that ADP-arsenate hydrolysis was nonenzymic. Enzymic hydrolysis of ADP-arsenate was too slow, compared with its rapid nonenzymic hydrolysis, to be detected. The first order rate constant for ADP-arsenate hydrolysis at pH 7.5, 30 degrees C, was determined to be greater than 5 min-1 and was estimated to be 70 min-1. These data confirm previous suggestions that arsenate "uncouples" oxidative phosphorylation by a mechanism involving synthesis of ADP-arsenate, followed by its rapid nonenzymic hydrolysis.

Demonstration and quantitation of catalytic and noncatalytic bound ATP in submitochondrial particles during oxidative phosphorylation.

Techniques are described for studying the labeling of ADP and ATP bound to the ATP synthase complex of beef heart submitochondrial particles catalyzing oxidative phosphorylation. These suffice for measurements of bound nucleotides during the time required for a single turnover, during steady state net ATP synthesis, or under quasiequilibrium conditions of ATP formation and hydrolysis. Results show that the "tightly bound" ATP associated with isolated submitochondrial particles does not become labeled by medium [32P]Pi rapidly enough to qualify as an intermediate in ATP synthesis. In contrast to chloroplast preparations, little or no bound [32P]Pi committed to ATP formation is present on particles during steady state synthesis. Also, highly active particles synthesizing ATP from [32P]Pi and filtered after EDTA addition have no detectable bound [32P]ATP even though several ATPs have been made per synthase complex. However, under quasiequilibrium conditions membrane-bound ADP and ATP are present whose labeling characteristics qualify them as intermediates in ATP synthesis. In addition, a hexokinase-accessibility approach shows the presence of a steady level of bound ATP. Lack of detection of bound intermediates under other conditions is regarded as reflecting the ready reversibility of oxidative phosphorylation, with consequent facile cleavage of bound ATP and release of bound Pi.

Interaction of ox heart mitochondrial F1-ATPase with immobilized ADP and ATP.

The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The column-bound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and mono-phosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro-7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking co-operative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.

Inhibition of phosphoglucomutase by vanadate.

Phosphoglucomutase is inhibited by a complex formed from alpha-D-glucose 1-phosphate (Glc-1-P) and inorganic vanadate (Vi). Both the inhibition at steady state and the rate of approach to steady state are dependent on the concentrations of both Glc-1-P and Vi. Inhibition is competitive versus alpha-D-glucose 1,6-bisphosphate (Glc-P2) and is ascribed to binding of the 6-vanadate ester of Glc-1-P (V-6-Glc-1-P) to the dephospho form of phosphoglucomutase (E). The inhibition constant for V-6-Glc-1-P at pH 7.4 was determined from steady-state kinetic measurements to be 2 x 10(-12) M. The first-order rate constant for approach to steady state increases hyperbolically with inhibitor concentration. The results are consistent with rapid equilibrium binding of V-6-Glc-1-P to E, with dissociation constant 1 x 10(-9) M, followed by rate-limiting conversion of the E.V-6-Glc-1-P complex to another species, E*.V-6-Glc-1-P, with first-order rate constant 4 x 10(-2)s-1. The rate constant determined for the reverse reaction, conversion of E*.V-6-Glc-1-P to E.V-6-Glc-1-P, is 2.5 x 10(-4)s-1. Formation of E*.V-6-Glc-1-P can also occur via binding of glucose 6-vanadate to the phospho form of phosphoglucomutase (E-P) followed by phosphoryl transfer and rearrangement of the enzyme-product complex.

1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of baculovirus-overexpressed cytosolic phospholipase A2 using a single-step affinity column chromatography.

Cytosolic phospholipase A2 (cPLA2) plays a key role in the production of proinflammatory lipid mediators such as prostaglandins, thromboxane, and leukotrienes. cPLA2, an arachidonic acid-selective, 85-kDa protein has been purified, cloned, and partially characterized from a number of tissues. However, the purification schemes previously published by several groups are lengthy, involving several chromatographic steps and resulting in low yields of enzyme. Here we report the preparation of a novel affinity column (Affi-656) by immobilizing a competitive inhibitor of cPLA2, and a single-step purification of this enzyme. This column selectively retains cPLA2 activity from the cytosolic fractions of Sf9 cells infected with recombinant baculovirus which is eluted by a gradient of CHAPS in the elution buffer. Purification of cPLA2 to homogeneity can thus be accomplished in a single step. Moreover, mutant cPLA2 (Ser505/Ala505) which is no longer phosphorylated at Ser505 is also retained on Affi-656; mutation at this residue does not disrupt its binding to the affinity column. To our knowledge, this is the first report of cPLA2 affinity purification. Affi-656 is a convenient, reproducible, and high-capacity affinity column, and is a valuable tool for rapid purification of cPLA2 in large quantities.