Showing papers on "Vanadate published in 1991"
TL;DR: This chapter discusses the use of vanadate as protein-phosphotyrosine phosphatase inhibitor, which is conveniently separable from the phosphoseryl and phosphothreonyl phosphatases activities inhibited by EDTA and fluoride.
Abstract: Publisher Summary This chapter discusses the use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Vanadium ions have found widespread use as an inhibitor of protein-phosphotyrosine phosphatases. Some form of vanadium ion is added to preserve the phosphotyrosine content of cells, cell lysates, and tyrosine kinase assays. Protein-phosphotyrosine phosphatase (PPTPase) activity is found in most mammalian and avian cells and tissues as well as in bacterial and yeast cultures. This uncertainty in substrate suitability may influence the effectiveness of inhibitors. The multiple phosphotyrosine phosphatases may be more or less sensitive to the same inhibitor. The protein-phosphotyrosine phosphatase activities sensitive to inhibition by zinc chloride and sodium orthovanadate are conveniently separable from the phosphoseryl and phosphothreonyl phosphatase activities inhibited by EDTA and fluoride.
585 citations
TL;DR: The Ca2+ pump-inhibitory sesquiterpene lactone, thapsigargin, is shown to distinguish these two Ca 2+ pools and identify a third Ca2- pumping pool unresponsive to InsP3 or GTP.
225 citations
TL;DR: It is concluded that proton excretion is mediated by an active proton pump on the apical membrane of the gill epithelium similar to that reported for the frog skin.
Abstract: Ion transport inhibitors, amiloride, SITS, vanadate and acetazolamide, wereadded to the water to determine the effect of ion transfer mechanisms on the acidification of water passing over the gills. In neutral water, proton excretion causes a marked reduction in gill water pH. If water pH is 2.5 units lower than blood pH, however, then this proton excretion is inhibited and all water pH changes can be accounted for by CO 2 hydration and ammonia protonation. Proton excretion across the gills is insensitive to 0.1mmoll −1 amiloride and SITS but sensitive to vanadate, acetazolamide and water pH; thus, we conclude that proton excretion is mediated by an active proton pump on the apical membrane of the gill epithelium similar to that reported for the frog skin. Higher concentrations of amiloride (0.5 and 1mmoll −1 ) reduced both ammonia and acid excretion, presumably because of inhibition of Na + /K + -ATPase on the basolateral border ofthe gill epithelium.
191 citations
TL;DR: The data indicate that vanadate is an effective oral hypoglycemic treatment in NIDDM states and suggest that its major effects occurs distal to the insulin receptor tyrosine kinase.
Abstract: We have studied the effects of oral administration of vanadate, an insulinometic agent and a potent inhibitor of phosphotyrosyl protein phosphatase (PTPase) in vitro, on blood glucose and PTPase action, in two hyperinsulinemic rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Oral administration of vanadate (0.25 mg/ml in the drinking water) to ob/ob mice for 3 wk lowered blood glucose level from 236 +/- 4 to 143 +/- 2 mg/dl without effect on body weight. Administration of vanadate to db/db mice produced a similar effect. Electron microscopic examination revealed no signs of hepatotoxicity after 47 d of treatment. There was a slight reduction in insulin receptor autophosphorylation when tested by immunoblotting with antiphosphotyrosine antibody after in vivo stimulation, and the phosphorylation of the endogenous substrate of the insulin receptor, pp185, was markedly decreased in the ob/ob mice. Both cytosolic and particulate PTPase activities in liver of ob/ob mice measured by dephosphorylation of a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation were decreased by approximately 50% (P less than 0.01). In db/db diabetic mice, PTPase activity in the cytosolic fraction was decreased to 53% of control values (P less than 0.02) with no significant difference in the particulate PTPase activity. Treatment with vanadate did not alter hepatic PTPase activity as assayed in vitro, or receptor and substrate phosphorylation as assayed in vivo, in ob/ob mice despite its substantial effect on blood glucose. These data indicate that vanadate is an effective oral hypoglycemic treatment in NIDDM states and suggest that its major effects occurs distal to the insulin receptor tyrosine kinase.
167 citations
TL;DR: The vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase and should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.
149 citations
Journal Article•
TL;DR: The high [Ca2+]i found in the tube tip with fura-2 seems to indicate the real Ca2+ concentration and is probably responsible for vesicle fusion, fragmentation of actin filaments, and inhibition of cytoplasmic streaming.
136 citations
TL;DR: Cytotoxicity, morphological transformation and cellular retention have been studied in BALB/3T3 Cl A 31-1-1 cells and corresponding gel-filtration experiments indicate that a redistribution of the element among the cytosol components occurs with time.
Abstract: Cytotoxicity, morphological transformation and cellular retention have been studied in BALB/3T3 Cl A 31-1-1 cells for ammonium or sodium vanadate [vanadium(V)] and for vanadyl sulphate [vanadium(IV)]. A morphological transformation focus assay showed transforming activity for vanadium(V) (P less than 0.005 at concentrations of 3 x 10(-6) or higher) while vanadium(IV) was not transforming in the cells. Cytotoxicity was higher for vanadium(V) than for vanadium(IV); this was particularly clear at doses from 5 x 10(-6) to 5 x 10(-5) M. The cellular retention of both vanadate and vanadyl compounds at 24, 48 and 72 h incubation was similar. At concentrations lower than 10(-6) M vanadate, the retention was linear with the dose, while at higher exposures the vanadium taken up by the cells levelled off or slightly decreased. Exposure to 10(-6) M and 10(-5) M vanadium(V) for 3 and 24 h as well as to 10(-6) M for 48 and 72 h yielded greater than 94% vanadium in the cytosol, but exposure to a toxic dose (10(-5) M) for 48 and 72 h yielded 20% vanadium associated with cellular organelles, which suggests that some sites in the cytosol become saturated with vanadium. The corresponding gel-filtration experiments indicate that a redistribution of the element among the cytosol components occurs with time.
94 citations
TL;DR: A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship and an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V( 4+-OOH.
Abstract: Vanadate induces phosphotyrosine accumulation and activates O2 consumption in permeabilized differentiated HL60 cells. NADPH, the substrate of the respiratory burst oxidase, was found to be necessary not only for the increased O2 consumption, but also for tyrosine phosphorylation. The effect of NADPH was not due to reduction of vanadate to vanadyl. Instead, NADPH was required for the synthesis of superoxide, which triggered the formation of peroxovanadyl [V(4+)-OO] and vanadyl hydroperoxide [V(4+)-OOH]. One or both of these species, rather than vanadate itself, appears to be responsible for phosphotyrosine accumulation and activation of the respiratory burst. Accordingly, the stimulatory effects of vanadate and NADPH were abrogated by superoxide dismutase. Moreover, phosphorylation was activated in the absence of NADPH by treatment with V(4+)-OO and/or V(4+)-OOH, generated by treatment of orthovanadate with KO2 or H2O2 respectively. The main source of the superoxide involved in the formation of V(4+)-OO and V(4+)-OOH is the NADPH oxidase. This was shown by the inhibitory effects of diphenylene iodonium and by the failure of undifferentiated cells, which lack oxidase activity, to undergo tyrosine phosphorylation when treated with vanadate and NADPH. By contrast, exogenously generated V(4+)-OO induced marked phosphorylation in the undifferentiated cells, demonstrating the presence of the appropriate tyrosine kinases and phosphatases. A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship. Therefore an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V(4+)-OOH. These peroxides promote phosphotyrosine formation, most likely by inhibition of tyrosine phosphatases. Accumulation of critical tyrosine-phosphorylated proteins then initiates a respiratory burst, with abundant production of superoxide. The newly formed superoxide catalyses the formation of additional V(4+)-OO and/or V(4+)-OOH, thereby magnifying the response. Since vanadium derivatives are ubiquitous in animal tissues, V(4+)-OO and/or V(4+)-OOH could be formed in vivo by reduced O2 metabolites, becoming potential endogenous tyrosine phosphatase inhibitors. Because of their potency, peroxides of vanadate may be useful as probes for the study of protein phosphotyrosine turnover.
89 citations
TL;DR: Considering the different effects of bafilomycin and vanadate on fluid and ion secretion a new working hypothesis for a transport model is proposed.
80 citations
TL;DR: It is demonstrated that K(+)-activated ATPase is present in apical membranes of rat distal colon and permit the speculation that this enzyme represents a unique and distinct ATPase and is likely linked closely to the active K+ absorptive process present in this epithelium.
Abstract: This study sought to establish the presence of K(+)-activated adenosinetriphosphatase (ATPase) activity in the colonic mucosa of the rat distal colon. K(+)-activated ATPase activity was present in apical membranes but not in basolateral membranes. K(+)-activated ATPase activity in apical membranes represented an approximate 10-fold enrichment compared with that in the homogenate. Na(+)-K(+)-activated ATPase activity was also present in homogenate but was enriched less than fourfold in apical membranes. K(+)-activated ATPase activity in apical membranes had both ouabain-sensitive and ouabain-insensitive components. In contrast, Na(+)-K(+)-activated ATPase activity was completely inhibited by ouabain. Similar half-maximal concentrations for K+ and pH activation curves were found for both ouabain-sensitive and ouabain-insensitive fractions. In addition to K+, the ouabain-sensitive fraction of K(+)-activated ATPase activity was stimulated by Rb+, NH+4, and Cs+, whereas the ouabain-insensitive fraction was activated only by Rb+. K(+)-activated ATPase activity was significantly inhibited by vanadate but not by N-ethylmaleimide or omeprazole. In the proximal colon, in contrast to the distal colon, active K+ absorption is not present, and K(+)-activated ATPase is approximately 20% of that in the distal colon. These studies demonstrate that K(+)-activated ATPase is present in apical membranes of rat distal colon and permit the speculation that this enzyme represents a unique and distinct ATPase (compared with either Na(+)-K(+)-ATPase or gastric parietal cell K(+)-ATPase) and is likely linked closely to the active K+ absorptive process present in this epithelium.
77 citations
TL;DR: Direct toxic effects due to a reduced flow rate contribute only partly to vanadate hepatotoxicity and do not affect mitochondrial integrity, and the inhibition of both processes in parallel by antioxidants are suggestive of a causative role for lipid peroxidation inVanadate-induced hepatot toxicity.
TL;DR: In this paper, the partial oxidation of ethane has been investigated on silica-supported alkali metal vanadate catalysts in a fixed-bed continuous-flow reactor at 770-823 K using N2O as oxidant.
TL;DR: The notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in Trypanosoma cruzi plasma membrane vesicles is supported.
Abstract: High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.
TL;DR: The data indicate the presence of at least three separate Ca2+ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP.
Abstract: Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP3) sensitive and IP3-insensitive nonmitochondrial intracellular Ca2+ pools in acinar cells of the exocrine pancreas. For further characterization of Ca2+ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designated P1 to P5 from the top to the bottom of the gradient. Measuring Ca2+ uptake and Ca2+ release with a Ca2+ electrode, we could differentiate three nonmitochondrial intracellular Ca2+ pools: (i) an IP3-sensitive Ca2+ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca2+ pool (CasCaP), vanadate- and IP3-insensitive, and (iii) a vanadate-sensitive Ca2+ pool (VasCaP), neither IP3- nor caffeine-sensitive, into which Ca2+ uptake is mediated via a Ca2+ ATPase sensitive to vanadate at 10(-4) mol/liter. A fourth Ca2+ pool is neither IP3- nor caffeine- or vanadate-sensitive. Percoll fraction P1 contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca2+ uptake and Ca2+ release. When membrane vesicles were incubated in the presence of caffeine (2 x 10(-2) mol/liter), Ca2+ uptake up to the steady state [Ca2+] did not appear to be altered as compared to the control Ca2+ uptake. However, in control vesicles spontaneous Ca2+ release occurred after the steady state had been reached, whereas caffeine-pretreated vesicles did not spontaneously release Ca2+. Addition of IP3 at steady state [Ca2+] induced similar Ca2+ release followed by Ca2+ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca2+ was released from all Ca2+ pools including the IsCaP. Following Ca2+ reuptake after IP3 had been added, a second addition of IP3 to control vesicles induced further but smaller Ca2+ release, and a third addition resulted in a steady Ca2+ efflux by which all Ca2+ that had been taken up was released. This steady Ca2+ release started at a Ca2+ concentration between 5.5-8 x 10(-7) mol/liter and could also be induced by the IP3 analogue inositol 1,4,5-trisphosphorothioate (IPS3) or by addition of Ca2+ itself. Ruthenium red (10(-5) mol/liter) inhibited both caffeine-induced as well as Ca2(+)-induced but not IP3-induced Ca2+ release. Heparin (100 micrograms/ml) inhibited IP3- but not caffeine-induced Ca2+ release. The data indicate the presence of at least three separate Ca2+ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca2+ uptake these Ca2+ pools appear to be separate.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: This investigation indicates that vanadate can normalize many of the metabolic abnormalities seen in the liver of genetically diabetic db/db mice, a model for non-insulin-dependent diabetes mellitus (NIDDM), which suggests a partial reversal of insulin resistance.
Abstract: The effect of long-term (12 weeks) oral treatment with sodium orthovanadate on hepatic glycogen metabolizing and lipogenic enzymes was studied in genetically diabetic db db mice. These mice were characterized by significant (P db db mice when compared with the nondiabetic ones. Vanadate caused significant decreases in phosphorylase a (P db db mice, a model for non-insulin-dependent diabetes mellitus (NIDDM). Vanadate also causes a decrease in plasma insulin level, along with normalization of plasma glucose, which suggests a partial reversal of insulin resistance.
TL;DR: The results suggest that a small number of carboxyl residues in the sodium pump alpha-subunit (perhaps one) are essential for K+ and Na+ binding and stabilizing the occluded enzyme cation forms.
TL;DR: The Na(+)/H(+) antiporter seems to play a principal role in internal pH regulation in Dunaliella and is suggested to be involved in intracellular acidification-induced change in the internal Na(+) concentration.
Abstract: Na+/H+ exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na+ were followed. Following a mild intracellular acidification, intracellular Na+ content increased dramatically and then decreased. We interpret the phase of Na+ influx as due to the activation of the plasma membrane Na+/H+ antiporter and the phase of Na+ efflux as due to an active Na+ extrusion process. The following observations are in agreement with this interpretation: (a) the Na+ influx phase was sensitive to Li+, which is an inhibitor of the Na+/H+ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na+ efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na+ concentration is due to a combination of Na+ uptake via the Na+/H+ antiporter and an active, ATPase-dependent, Na+ extrusion. The Na+/H+ antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.
TL;DR: It was suggested that another mechanism distinct from myosin light chain phosphorylation might be involved in Ca2(+)-independent contraction of uterine smooth muscle elicited by oxytocin.
TL;DR: Despite cross-reacting with antibodies against the cardiac and skeletal isoforms, the cerebellar microsomal Ca(2+)-ATPase appears to be distinct from both muscle enzymes.
Abstract: The microsomal fraction of chicken cerebellum expresses a large amount of Ca(2+)-ATPase (105 kDa), which is phosphorylated by ATP in the presence of Ca2+. The Ca(2+)-ATPase activity is highly sensitive to temperature and to the presence of detergents. This ATPase has kinetic properties similar to those of chicken skeletal-muscle sarcoplasmic reticulum, as (i) it is activated by low (microM) and inhibited by high (mM) Ca2+ concentrations, (ii) it shows biphasic activation with ATP and (iii) it is inhibited by vanadate. However, the vanadate-sensitivity is at least 10 times greater than that observed in chicken skeletal or cardiac sarcoplasmic-reticulum Ca(2+)-ATPases. Thus, despite cross-reacting with antibodies against the cardiac and skeletal isoforms, the cerebellar microsomal Ca(2+)-ATPase appears to be distinct from both muscle enzymes. The Ca(2+)-ATPase is concentrated in, but not exclusive to, Purkinje neurons. In Purkinje neurons the Ca(2+)-ATPase appears to be expressed throughout the cell body, the dendritic tree (and the spines) and the axons. At the electron-microscope level the Ca(2+)-ATPase is found in smooth and rough endoplasmic-reticulum cisternae as well as in other, yet unidentified, smooth-surfaced structures.
TL;DR: Data suggest a coupling between the charge-transfer and ATP hydrolysis domains and establish a solid basis for future probing of the electrogenic properties of the yeast H(+)-ATPase.
TL;DR: The results here reported suggest that chronic administration of vanadyl or vanadate in the drinking water is not a viable alternative treatment to insulin in human diabetes.
Abstract: Vanadate and vanadyl, two forms of vanadium, have been reported to exert insulin-like effects in vivo and in vitro. In the present study we compared the effectiveness of oral sodium metavanadate (NaVO3), sodium orthovanadate (Na3VO4) and vanadyl sulphate pentahydrate (VOSO4.5H2O) treatment in alleviating some signs of diabetes in streptozotocin-induced diabetic rats. Vanadium compounds were administered in aqueous solutions of NaCl (80 mM) at concentrations of 0.20 mg/ml (NaVO3), 0.50 mg/ml (Na3VO4), and 1.1 mg/ml (VOSO4.5H2O) for two weeks. Control rats, either diabetic or non-diabetic, drank solutions of NaCl (80 mM). Although some signs of diabetes (hyperglycaemia, hyperphagia, polydipsia) were significantly ameliorated by the vanadium treatment, negative side effects were also observed in all of the vanadium-treated diabetic rats. Those effects included some deaths, decreased weight gain, and tissue vanadium accumulation, which are consistent with the reported toxicity of vanadium in non-diabetic rats. Vanadyl sulphate was the most effective compound of those tested in normalizing blood glucose levels. However, the results here reported suggest that chronic administration of vanadyl or vanadate in the drinking water is not a viable alternative treatment to insulin in human diabetes.
TL;DR: The condensation reactions occurring between vanadate and a number of amino acids and simple peptides have been studied by 51V nuclear magnetic resonance (NMR) signals as discussed by the authors.
Abstract: The condensation reactions occurring between vanadate and a number of amino acids and simple peptides have been studied by 51V nuclear magnetic resonance. Vanadate and ligand stoichiometry has been established for the complexes formed and their formation constants determined. The amino acids were all found to undergo rather weak interactions with vanadate and in general provided two products with 51V chemical shifts near −545 and −555 ppm. The peptides also gave minor products with NMR signals occurring with that of vanadate itself. However, in this case, the major products occur at approximately −510 ppm. These latter complexes are monovanadate, monoligand complexes which require the terminal amino, the carboxylate groups and an unsubstituted nitrogen in the peptide linkage in order for product formation to occur. Sidechains promote product formation, apparently by favouring a more readily complexed peptide conformation. Formation constants vary from about 20 to 50 M−1 depending on the sidechain. The car...
TL;DR: Measurements of K+ efflux revealed two apparent sites for activation by PTX, consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.
Abstract: Palytoxin (PTX), isolated from the marine soft coral Palythoa tuberculosa, increases the cation conductance of human red cell membranes. In the presence of 10(-10) M PTX and 10(-5) M DIDS, the membrane potential approximates the equilibrium potential for Na+ or K+ rather than Cl-. Even in the absence of DIDS, the Na+ and K+ conductances were greater than the Cl- conductance. The selectivity of the PTX-induced cation conductance is K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ much greater than choline+ greater than TEA+ much greater than Mg2+. Measurements of K+ efflux revealed two apparent sites for activation by PTX, one with a Kal of 0.05 nM and a maximum flux, nu max1, of 1.4 mol/liter of cells per h and another with a Ka2 of 98 nM and a nu max2 of 24 mol/liter of cells per h. These effects of PTX are completely blocked by external ouabain (300 microM) and prevented by internal vanadate (100 microM). When the PTX channels are open, the Na,K pumps do not catalyze ATP hydrolysis. Upon thorough washout of cells exposed to about five molecules of PTX/pump, the Na,K pump of these cells operates normally. Blockage of the positively charged NH2 terminus of PTX with a p-bromobenzoyl group reduces the potency of the compound to induce Na and K fluxes by at least a factor of 100, and to compete with the binding of [3H]ouabain by at least a factor of 10. These data are consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.
TL;DR: The rate and mechanism of entry after completely inactivating the pump were examined and it was found that passive Ca2+ movements occur predominantly by a reversible carrier-mediated mechanism for which Sr2+ is an alternate substrate.
Abstract: The intracellular Ca2+ concentration of nearly all cells is kept at submicromolar levels. The magnitudes of transmembrane Ca2+ movement that maintain this steady state in the human red blood cell have long been debated. Although there is agreement that the physiologic extrusion of Ca2+ by the well-characterized Ca2+. ATPase amounts to 45 mumol/liter cells per h (1982. Nature (Lond.). 298:478-481), the reported passive entry rates in physiological saline (2-20 mumol/liter cells per h) are all substantially lower. This discrepancy could be due to incomplete inhibition of the pump in the previous measurements of Ca2+ entry. We therefore examined both rate and mechanism of entry after completely inactivating the pump. This required pretreatment with iodoacetamide (to lower the intracellular ATP concentration) and vanadate (to inhibit any residual Ca2+ pump activity). The rate of Ca2+ entry (53 mumol/liter cells per h) was now found to be comparable to the accepted extrusion rate. Entry closely obeyed Michaelis-Menten kinetics (Vmax = 321 +/- 17 nmol Ca/g dry wt per h, Km = 1.26 +/- 0.13 mM), was competitively inhibited by external Sr2+ (Ki = 10.8 +/- 1.2 mM), and was accelerated by intracellular Ca2+. 45Ca2+ efflux from these pump-inactivated cells was also accelerated by either external Ca2+ or Sr2+. These accelerating effects of divalent cations on the opposite (trans) face of the membrane rule out a simple channel. Substrate-gated channels are also ruled out: cells equilibrated with 45Ca2+ lost the isotope when unlabeled Ca2+ or Sr2+ was added externally. Thus, passive Ca2+ movements occur predominantly by a reversible carrier-mediated mechanism for which Sr2+ is an alternate substrate. The carrier's intrinsic affinity constants for Ca2+ and Sr2+, 1.46 and 0.37 mM-1, respectively, indicate that Ca2+ is the preferred substrate.
TL;DR: An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
Abstract: In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.
TL;DR: The pma2 gene of Schizosaccharomyces pombe codes for a polypeptide having a predicted Mr of 110,126 and which is 79% identical to the plasma membrane H(+)-ATPase encoded by the pma1 gene, demonstrating that the two H(+)-ATPases are functionally interchangeable in vivo but differently expressed.
TL;DR: It is shown that peroxovanadates are useful and important agents for investigating the mechanism of action of insulin in skeletal muscle and are maximally stimulated the rate of glycogen synthesis in incubated soleus muscles isolated from Wistar rats.
Abstract: 1. The insulin-like effects of orthovanadate (10 mM) and peroxides of vanadate (peroxovanadates, at 1 mM) on rates of lactate formation, glucose oxidation and glycogen synthesis were measured in incubated soleus-muscle preparations isolated from non-obese Wistar rats and lean (fa/?) or insulin-resistant obese Zucker (fa/fa) rats. 2. The stimulation of the rates of lactate formation and glucose oxidation by either orthovanadate or peroxovanadates was of similar magnitude to the stimulation by a maximally effective concentration of insulin (1000 microunits/ml). 3. Peroxovanadates, but not orthovanadate, maximally stimulated the rate of glycogen synthesis in incubated soleus muscles isolated from Wistar rats. 4. When soleus-muscle preparations were incubated in the presence of both insulin (1000 microunits/ml) and peroxovanadates (1 mM), this did not result in a synergistic increase in the rate of total glucose utilization as compared with either agent alone. 5. Soleus muscles isolated from obese (fa/fa) Zucker rats exhibited a decrease in response to a physiologically relevant concentration of insulin (100 microunits/ml). Peroxovanadates, at 1 mM, maximally stimulated the rate of glycogen synthesis in soleus muscles isolated from obese (fa/fa) Zucker rats. 6. The findings indicate that peroxovanadates are useful and important agents for investigating the mechanism of action of insulin in skeletal muscle.
TL;DR: The results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect, and an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially preventedVanadate inhibition of stomatal opening.
Abstract: An H+ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K+ and Cl− uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H+-ATPase inhibitor, vanadate, in inhibiting both H+ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations≥50 mM. In contrast, in the absence of Cl−, when V2O5 was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K+ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH2CO2H)2). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid, 1 mM 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, 200 μM Zn2+) were not effective. Decreased vanadate inhibition at high Cl−/vanadate ratios may result from competition between vanadate and Cl− for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H+ ATPase.
TL;DR: The results demonstrate the presence of a primary Ca2+ transport ATPase in the plasma membrane of wheat roots and confirms that the ATP-binding site of the 45Ca2- transport system, similar to the H+-ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane.
Abstract: Plasma membrane preparations of high purity were obtained from roots of dark-grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. These preparations mainly contained sealed, right-side-out vesicles (ca 90% exposing the original outside out). By subjecting the preparations to 4 freeze/thaw cycles the proportion of sealed, inside-out (cytoplasmic side out) vesicles increased to ca 30%. Inside-out and right-side-out plasma membrane vesicles were then separated by partitioning the freeze/thawed plasma membranes in another aqueous polymer two-phase system. In this way, highly purified, sealed, inside-out (>60% inside-out) vesicles were isolated and subsequently used for characterization of the Ca2+ transport system in the wheat plasma membrane. The capacity for 45Ca2+ accumulation, nonlatent ATPase activity and proton pumping (the latter two markers for inside-out plasma membrane vesicles) were all enriched in the inside-out vesicle fraction as compared to the right-side-out fraction. This confirms that the ATP-binding site of the 45Ca2+ transport system, similar to the H+-ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The 45Ca2+ uptake was MgATP-dependent with an apparent Km for ATP of 0.1 mM and a high affinity for Ca2+ [Km(Ca2+/EGTA) = 3 μM]. The pH optimum was at 7.4–7.8. ATP was the preferred nucleotide substrate with ITP and GTP giving activities of 30–40% of the 45Ca2+ uptake seen with ATP. The 45Ca2+ uptake was stimulated by monovalent cations; K− and Na+ being equally efficient. Vanadate inhibited the 45Ca2+ accumulation with half-maximal inhibitions at 72, 57 and 2 μM for basal, total (with KCI) and net K+-stimulated uptake, respectively. The system was also highly sensitive to erythrosin B with half-maximal inhibition at 25 nM and total inhibition at 1μM. Our results demonstrate the presence of a primary Ca2+ transport ATPase in the plasma membrane of wheat roots. The enzyme is likely to be involved in mediating active efflux (ATP-binding sites on the cytoplasmic side) to the plant cell exterior to maintain resting levels of cytoplasmic free Ca2+ within the cell.