The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.     

Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.     

Vanadate binding to the gastric H,K-ATPase and inhibition of the enzyme's catalytic and transport activities

Vanadate inhibition of the catalytic and transport activities of the gastric magnesium-dependent, hydrogen ion transporting, and potassium-stimulated adenosinetriphosphatase (EC 3.6.1.3) (H,K-ATPase) has been studied. The principal experiment observations are the following: (1) Inhibition of adenosine 5'-triphosphate (ATP) hydrolysis is biphasic. Vanadate binding with a stoichiometry of 1.5 nmol mg-1 approximately halves K+-stimulated ATPase activity at physiological temperature. The remaining activity is inhibited by binding an additional 1.5 nmol mg-1 vanadate with lower apparent ions bind specifically to gastric vesicles with two affinities. Vanadate binding in the presence of nucleotide is compatible with competition for the kinetically defined high-affinity and low-affinity ATP sites. (3) Vanadate inhibits phosphoenzyme formation and the K+-stimulated p-nitrophenyl phosphatase activity of the enzyme monophasically. A maximum of 1.5 nmol mg-1 acid-stable phosphoenzyme is formed. The half-time for vanadate dissociation from the site that inhibits p-nitrophenyl phosphate hydrolysis is 5 min (4) At most, 3 nmol mg-1 vanadate is required to inhibit proton transport. The simplest interpretation of the data is that vanadate inhibits the H,K-ATPase by binding competitively with ATP at two catalytic sites. Different catalytic mechanisms at the high-affinity and low-affinity sites are suggested by the different stoichiometries found for vanadate binding and phosphoenzyme formation.     

Inhibition and relaxation of sea urchin sperm flagella by vanadate

Direct measurements of the stiffness (elastic bending resistance) of demembranated sera urchin sperm flagella were made in the presence of MgATP2- and vanadate. Under these conditions, the flagellum is in a relaxed state, with a stiffness of approximately 0.9 x 10(-21) N m2, which is approximately 5% of the stiffness obtained in the rigor state in the absence of MgATP2-. MgADP- dose not substitute for MgATP2- in producing relaxed state. A progressive inhibition of movement is observed after addition of MgATP2- to flagella preincubated with vanadate, in which new bend generation, propagation, and relaxation by straightening are distinguished, depending on the ratio of MgATP2- and vanadate. At appropriate concentrations of vanadate, increase of the velocity of bend propagation is observed at a very low concentration of MgATP2- that is not enough to induce spontaneous beating. Vanadate enhances competitive inhibition of beat frequency by MgADP- but not by ADP3-, ATP4-, or Pi. These observations, and the uncompetitive inhibition of beat frequency by vanadate, indicate that vanadate can only bind to dynein-nucleotide complexes induced by MgATP2- and MgADP-. The state accessible by MgATP2- binding must be a state in which the cross-bridges are detached and the flagellum is relaxed. The state accessible by MgADP- binding must be a cross-bridged state. Bound vanadate prevents the transition between these two states. Inhibition and relaxation by banadate in the presence of MgATP2- results from the specific affinity of vanadate for a state in which nucleotide is bound, rather than a specific affinity for the deteched state.     

Interaction of vanadate with the cloned beta cell K(ATP) channel.

Vanadate is used as a tool to trap magnesium nucleotides in the catalytic site of ATPases. However, it has also been reported to activate ATP-sensitive potassium (K(ATP)) channels in the absence of nucleotides. K(ATP) channels comprise Kir6.2 and sulfonylurea receptor subunits (SUR1 in pancreatic beta cells, SUR2A in cardiac and skeletal muscle, and SUR2B in smooth muscle). We explored the effect of vanadate (2 mM), in the absence and presence of magnesium nucleotides, on different types of cloned K(ATP) channels expressed in Xenopus oocytes. Currents were recorded from inside-out patches. Vanadate inhibited Kir6.2/SUR1 currents by approximately 50% but rapidly activated Kir6.2/SUR2A ( approximately 4-fold) and Kir6. 2/SUR2B ( approximately 2-fold) currents. Mutations in SUR that abolish channel activation by magnesium nucleotides did not prevent the effects of vanadate. Studies with chimeric SUR indicate that the first six transmembrane domains account for the difference in both the kinetics and the vanadate response of Kir6.2/SUR1 and Kir6. 2/SUR2A. Boiling the vanadate solution, which removes the decavanadate polymers, largely abolished both stimulatory and inhibitory actions of vanadate. Our results demonstrate that decavanadate modulates K(ATP) channel activity via the SUR subunit, that this modulation varies with the type of SUR, that it differs from that produced by magnesium nucleotides, and that it involves transmembrane domains 1-6 of SUR.     

Interaction of vanadate with membrane-bound ATPase from Mycobacterium phlei

Vanadate inhibited the formation of proton gradient and membrane potential as well as Ca2+ transport by everted membrane vesicles from Mycobacterium phlei, with half-maximal inhibition occurring at 5 to 14 microM. That this is due to the inhibition of the proton-translocating ATPase was suggested by the observation that the inhibition described above occurred only when the processes were driven by the hydrolysis of ATP but not when energized by the oxidation of succinate and NADH. Furthermore, vanadate did indeed inhibit ATP hydrolysis by these membrane vesicles. Although the inhibition of ATP hydrolysis could be demonstrated only in the presence of high concentrations (e.g. 11 mM) of Mg2+, this was presumably due to the fact that we were measuring the sum of ATP hydrolysis by both coupled and partially uncoupled enzymes. This is the first reported effect of vanadate on bacterial proton-translocating ATPase.     

The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions (Simons, T. J. B. (1979) Nature 281, 337-338). Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C (Weigel, P. H., and Oka, J. A. (1983) J. Biol. Chem. 258, 5089-5094); vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.     

Relationship of major phosphorylation reactions and MgATPase activities to ATP-dependent shape change of human erythrocyte membranes

Human erythrocyte ghosts prepared by hemolysis and washing in hypotonic Tris are crenated by salt and divalent cations, but undergo shape change to smooth biconcave discs and stomatocytic forms when incubated with MgATP at 37 degrees C. This is normally accompanied by protein and lipid phosphorylations in which the major phosphate acceptors are the spectrin beta-chain and inositol phospholipids, respectively. The system was manipulated in several ways to demonstrate the independence of ATP-dependent shape change from the major phosphorylation reactions. Salt-extracted membranes incubated with adenosine, an inhibitor of spectrin and phosphatidylinositol kinases, underwent normal shape change despite reductions of greater than 90% in spectrin and phospholipid labeling by [gamma-32P]ATP. ATP-dependent shape change was blocked by vanadate at micromolar concentrations (half-maximal inhibition at less than 1 microM), but vanadate did not inhibit membrane autophosphorylation reactions or turnover of spectrin- or lipid-bound phosphate. Vanadate inhibited part of the ATP hydrolysis that accompanies shape change and is expressed in the presence of ouabain and EGTA. The vanadate-sensitive MgATPase activity was approximately 3 nmol Pi X min-1 X mg of protein-1. The results implicate it in ATP-dependent shape change.     

Vanadate: a potent inhibitor of multifunctional glucose-6-phosphatase

Vanadate has been found to be a potent inhibitor of both the hydrolytic and synthetic activities of the multifunctional enzyme glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). The enzyme, when studied in both microsomal preparations and in situ using permeable isolated hepatocytes, is inhibited by micromolar concentrations of vanadate. The inhibition by vanadate is greater in detergent-treated than in untreated microsomes. In both the microsomal preparations and permeable hepatocytes, the inhibition by vanadate is competitive with the phosphate substrate and is greater for the phosphotransferase than the hydrolase activity of the enzyme. The Ki values of vanadate for carbamyl-phosphate : glucose phosphotransferase and glucose-6-phosphate phosphohydrolase determined with permeable hepatocytes are in good agreement with the values determined with detergent-dispersed microsomes. The previously described inhibition of glucose-6-phosphate phosphohydrolase by ATP (Nordlie, R.C., Hanson, T.L., Johns, P.T. and Lygre, D.G. (1968) Proc. Natl. Acad. Sci. USA 60, 590-597) can now be explained by the vanadium contamination of the commercially available ATP samples used. In contrast with glucose-6-phosphatase, hepatic glucokinase and hexokinase were not inhibited by vanadate. Physiological implications and utilitarian experimental applicability of vanadate as a selective metabolic probe, based on these observations, are suggested.     

Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 μm. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 μM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and “banana peel” structures in which tubules have peeled back from the axoneme. Addition of 5 μM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 μM ATP causes resumption of flagellar beating. Addition of 40 μM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 μM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.     

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum vesicles was studied in a native and a fluorescein-labeled ATPase preparation (Pick, U., and Karlish, S. J. D. (1980) Biochim. Biophys. Acta 626, 255-261). Vanadate induced a fluorescence enhancement in a fluorescein-labeled enzyme, indicating that it shifts the equilibrium between the two conformational states of the enzyme by forming a stable E2-Mg-vanadate complex (E2 is the low affinity Ca2+ binding conformational state of the sarcoplasmic reticulum Ca-ATPase). Indications for tight binding of vanadate to the enzyme (K1/2 = 10 microM) in the absence of Ca2+ and for a slow dissociation of vanadate from the enzyme in the presence of Ca2+ are presented. The enzyme-vanadate complex was identified by the appearance of a time lag in the onset of Ca2+ uptake and by a slowing of the fluorescence quenching response to Ca2+. Ca2+ prevented the binding of vanadate to the enzyme. Pyrophosphate (Kd = 2 mM) and ATP (Kd = 25 microM) competitively inhibited the binding of vanadate, indicating that vanadate binds to the low affinity ATP binding site. Binding of vanadate inhibited the high affinity Ca2+ binding to the enzyme at 4 degrees C. Vanadate also inhibited the phosphorylation reaction by inorganic phosphate (Ki = 10 microM) but had no effect on the phosphorylation by ATP. It is suggested that vanadate binds to a special region in the low affinity ATP binding site which is exposed only in the E2 conformation of the enzyme in the absence of Ca2+ and which controls the rate of the conformation transition in the dephosphorylated enzyme. The implications of these results to the role of the low affinity ATP binding sites are discussed.     

Effects of vanadate on Na+,K+-ATPase and on the force of contraction in guinea-pig hearts.

Vanadate has been shown to be a potent inhibitor of isolated Na⁺,K⁺-ATPase. Since the inhibition of this enzyme system has been implicated in a mechanism for the positive inotropic action of cardiac glycosides, the cardiac actions of vanadate were examined in connection with its action on Na⁺,K⁺-ATPase. Vanadate inhibited isolated Na⁺,K⁺-ATPase obtained from various tissues. The differences in the vanadate sensitivity due to enzyme source were relatively small. K⁺-stimulated phosphatase activity was more sensitive than Na⁺,K⁺-stimulated ATP hydrolysis. The compounds was more potent than phosphate in supporting [³H] oubain binding in the presence of Mg²⁺, indicating a higher affinity of the enzyme for vanadate. It, however, failed to inhibit oubain sensitive ⁸⁶Rb uptake in electrically stimulated atrial muscle of guinea-pig hearts in concentrations which would inhibit isolated Na⁺,K⁺-ATPase. These latter concentrations of vanadate also failed to produce positive inotropic effects in electrically stimulated left atrial preparations of guinea-pig hearts. Higher concentrations produced marked negative inotropic effects associated with a shortening of the action potential duration. These results indicate that vanadate is a potent inhibitor of isolated Na⁺,K⁺-ATPase, but cannot inhibit the enzyme in intact myocardial cells or produce positive inotropic effects when applied extracellularly. Inhibitory sites on the enzyme are probably located at the internal surface of the cell membrane which are normally inaccessible to vanadate in intact tissue.     

Vanadate: A potent inhibitor of multifunctional glucose-6-phosphatase

Vanadate has been found to be a potent inhibitor of both the hydrolytic and synthetic activities of the multi- functional enzyme glucose-6-phosphatase (d-glucose-6-phosphatase phosphohydrolase, EC 3.1.3.9). The enzyme, when studied in both microsomal preparations and in situ using permeable isolated hepatocytes, is inhibited by micromolar concentrations of vanadate. The inhibition by vanadate is greater in detergent-treated than in untreated microsomes. In both the microsomal preparations and permeable hepatocytes, the inhibition by vanadate is competitive with the phosphate substrate and is greater for the phosphotransferase than the hydrolase activity of the enzyme. The K_I values of vanadate for carbamyl-phosphate : glucose phosphotransferase and glucose-6-phosphate phosphohydrolase determined with permeable hepatocytes are in good agreement with the values determined with detergent-dispersed microsomes. The previously described inhibition of glucose-6-phosphate phosphohydrolase by ATP (Nordlie, R.C., Hanson, T.L., Johns, P.T. and Lygre, D.G. (1968) Proc. Natl. Acad. Sci. USA 60, 590–597) can now be explained by the vanadium contamination of the commercially available ATP samples used. In contrast with glucose-6-phosphatase, hepatic glucokinase and hexokinase were not inhibited by vanadate. Physiological implications and utilitarian experimental applicability of vanadate as a selective metabolic probe, based on these observations, are suggested.     

Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres.

In muscle fibres labelled with iodoacetamidotetramethylrhodamine at Cys707 of the myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power stroke, we monitored tension and linear dichroism of the probes in single glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by laser pulse photolysis of caged ATP and caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of ATP from caged ATP in the presence of calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-ATP) upon release of ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an ATP-containing relaxing solution to a rigor medium lacking ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of ATP. Neither adding nor removing ADP produced substantial changes in force. These results demonstrate that orientation of the rhodamine probes on the myosin head reflects mainly structural changes linked to nucleotide binding and release, rather than rotation of the cross-bridge during force generation.     

Effects of vanadate on the plasma membrane ATPase of red beet and corn

The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The K_i for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K⁺, and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the K_m and V_max of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats.     

Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers.

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.     

51V NMR study of vanadate binding to myosin and its subfragment 1

The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predominant vanadate form was monomeric, while at high concentration, it was tetrameric. The presence of myosin or S-1 in the solution produced a significant broadening of the signal of each form of vanadate, indicating that all of them bind to the protein. Addition of ATP, which does not affect the 51V NMR spectra in the absence of proteins, causes their significant alteration in the presence of myosin or S-1. The changes, which include the broadening of the signal of the monomeric and the narrowing of the signal of the oligomeric vanadate forms, indicate that more monomeric and less oligomeric vanadate binds to the proteins in the presence than in the absence of ATP. Irradiation by near-UV light in the presence of vanadate cleaves S-1 at three specific sites--at 23, 31, and 74 kDa from the N-terminus. The cleavages at 23 and 31 kDa are specifically inhibited by the addition of ATP. The vanadate-associated photocleavage of S-1 also depends on the total concentration of vanadate; it is observed only when the concentration of vanadate is at least 0.2 mM. This was also the lowest concentration at which oligomeric vanadate was detected in the 51V NMR spectra. From the parallel concentration dependence of the photocleavage and the appearance of the tetrameric vanadate, it is concluded that photocleavage occurs only when tetrameric vanadate binds to S-1.     

Vanadate stimulation of insulin release in normal mouse islets.

The effects of vanadate (Na3VO4) on pancreatic B-cell function were studied in normal mouse islets. Vanadate did not affect basal insulin release but potentiated the effect of 7-30 mM glucose at concentrations of 0.1-1 mM. This effect was progressive and slowly reversible. It was abolished by omission of extracellular Ca2+ but unaffected by blockers of adrenergic or muscarinic receptors. Comparison of the changes in membrane potential, 86Rb efflux and 45Ca efflux that vanadate and ouabain produced in B-cells made it possible to exclude the hypothesis that vanadate increases insulin release by blocking the sodium pump. Vanadate was also without effect on cAMP levels. On the other hand, it markedly changed the characteristics of the Ca(2+)-dependent electrical activity and of the oscillations of cytoplasmic Ca2+ recorded in B-cells stimulated by 15 mM glucose. In the steady state, Ca2+ influx was increased by vanadate, and this resulted in a rise in cytoplasmic Ca2+. The exact mechanisms underlying these changes could not be established but a blockade of K channels was excluded. In the presence of LiCl, vanadate markedly increased inositol phosphate levels in islet cells. This effect was attenuated but not suppressed by omission of Ca2+. A small increase in inositol bisphosphate was still produced by vanadate in the absence of LiCl. These results suggest that vanadate both stimulates phosphoinositide breakdown and inhibits inositol phosphate degradation. In conclusion, vanadate does not induce insulin release, but markedly potentiates the stimulation by glucose. This property is not due to an inhibition of the sodium pump or to a rise in cAMP concentration. It results from a complex interplay between changes in B-cell membrane potential, phosphoinositide metabolism and Ca2+ handling.     

Pertussis toxin-insensitive regulation of phosphatidylinositol hydrolysis by vanadate in brain microvessels.

Vanadate, over a concentration range from 0.1 to 0.5 mM, stimulated the incorporation of (32P)-orthophosphate into PI and PA in brain microvessels. At concentrations higher than 0.5 mM, the stimulatory effect of vanadate decreased. Concommitantly, an enhanced DAG production was observed, indicating that vanadate stimulated PI turnover. All these effects were evident at all the times tested. Experiments performed in the presence of pertussis toxin (IAP) indicated that a IAP-sensitive G-protein does not mediate the vanadate stimulated PI effect in brain microvessels.