Showing papers on "Vanadate published in 1981"
TL;DR: The purified and the reconstituted forms of the enzyme displayed the properties expected of the intact Ca2+ pump; they had an appropriate (Ca2+-Mg2+)-ATPase activity which displayed a relatively low affinity for Ca2+.
444 citations
TL;DR: The Calmodulin-depleted Ca2+-ATPase has been solubilized from the sarcolemmal membrane and "purified" on a calmodulin affinity chromatography column, and the major protein band has Ca2-dependent ATPase activity and can be phosphorylated by [gamma]32P]ATP in a Ca2+)-dependent reaction.
423 citations
TL;DR: This chapter highlights physiological and metabolic effects on enzyme activities of vanadium, affecting the transition state of phosphate, which seems to have an affinity for the binding sites on these enzymes.
Abstract: Publisher Summary This chapter highlights physiological and metabolic effects on enzyme activities. Vanadium, a group V element, belongs to the first transition series and can form compounds mainly in valence states 3+, 4+, and 5+, both anionic and cationic species. Several criteria of essentiality of an element are satisfied by the properties of vanadium such as low molecular weight, excellent catalytic activity, appropriate atomic structure, its position as a transition metal, ability to form chelates potentially with biologically active compounds, ubiquity in the geosphere and possibly in the biosphere, homeostatic regulation by controlled accumulation and rapid excretion, deficiency in animals and plants showing characteristic symptoms, and low toxicity on oral intake. At least three significant enzymes of the plasma membrane are affected by vanadate—Na, K-ATPase is inhibited in nanomolar concentrations and adenylate cyclase and NADH oxidase are activated in micro-molar to milli-molar concentrations. These enzymes are interrelated. ATP is the substrate for the first two, and both substrates are nucleotides. Vanadate, affecting the transition state of phosphate, seems to have an affinity for the binding sites on these enzymes.
157 citations
TL;DR: (Na,K)ATPase from kidney membranes has been reconstituted into proteoliposomes following solubilization in cholate, by the freeze—thaw sonication procedure described by Kasahara & Hinkle (1977).
Abstract: 1. (Na,K)ATPase from kidney membranes has been reconstituted into proteoliposomes following solubilization in cholate, by the freeze-thaw sonication procedure described by Kasahara & Hinkle (1977). The method is rapid and convenient.2. Upon addition of ATP to the exterior medium the reconstituted vesicles sustain high rates of active (22)Na uptake and (86)Rb efflux with many properties similar to those of the Na/K pump in well characterized cells such as erythrocytes.3. Observations on both active and passive transport of (22) Na and (86)Rb indicate that the vesicle population is heterogeneous; about 40 per cent contain Na/K pumps and the remainder seem to be plain lipid vesicles.4. The major Na(+)- or K(+)-stabilized non-phosphorylated conformational forms of the (Na, K)ATPase, E(1). Na and E(2). (K) respectively, have been investigated in the proteoliposomes, with particular regard to sidedness of the actions of Na(+) and K(+).5. Tryptic digestion of the vesicles reveals the Na(+)- and K(+)-stabilized conformations E(1). Na and E(2). (K) as characterized originally for purified (Na, K)ATPase (Jorgensen, 1975). Controlled trypsinolysis of Tris(+)-loaded vesicles in a Na(+)-or K(+)-containing medium leads to typical biphasic (Na(+)) or simple exponential (K(+)) time courses respectively, for loss of ATP-dependent (22)Na uptake (assayed subsequent to the tryptic digestion in the presence of inophores valinomycin plus FCCP). Tryptic digestion of K(+)- or Rb(+)- or Tris(+)-loaded vesicles suspended in a Na(+) medium results only in the biphasic (E(1). Na) pattern of loss of ATP-dependent (22)Na uptake.6. ATP-dependent (22)Na uptake and (86)Rb efflux are reduced by about the same extent following a short tryptic digestion in a Na(+)-containing medium.7. Vanadate ions inhibit ATP-dependent (22)Na uptake into the vesicles, at low concentrations (K(0.5) approximately 2 x 10(-7)m), following pre-incubation together with Mg(2+) and K(+) ions. K(+) ions in the medium are effective, K(+) ions within the vesicle are not. Na(+) ions in the medium prevent inhibition by vanadate+Mg(2+) but do not reverse inhibition in vesicles pre-incubated with vanadate, Mg(2+) and K(+) ions.8. The results show that the conformational forms E(1). Na and E(2). (K) are stabilized by Na(+) or K(+) ions respectively, bound to sites on the Na/K pump normally facing the cytoplasm. The significance of this finding is discussed in relation to the cation transport function of the pump.
139 citations
TL;DR: The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.
134 citations
TL;DR: Micromolar concentrations of vanadate added to quiescent human fibroblasts stimulate thymidine incorporation into DNA and produce an increase in cell numbers andVanadate also potentiated the mitogenic activity of fibroblast growth factor.
117 citations
TL;DR: The rate of NADH oxidation with oxygen as the acceptor is very low in mouse liver plasma membrane and erythrocyte membrane, but when vanadate is added, this rate is stimulated 10- to 20-fold, indicating that oxygen is the electron acceptor and the reaction is mediated by superoxide.
106 citations
TL;DR: In this paper, the properties of several ATPases present in synaptic membrane preparations from the cerebral cortex of rat were investigated, including the intrinsic (Na+ + K+)-ATPase and a low level of contaminating Mg2+-ATPases of mitochondrial origin, both of which could be controlled by the addition of ouabain and azide, respectively.
Abstract: We have investigated the properties of several ATPases present in synaptic membrane preparations from the cerebral cortex of rat. In addition to the intrinsic (Na+ + K+)-ATPase and a low level of contaminating Mg2+-ATPase of mitochondrial origin, both of which could be controlled by the addition of ouabain and azide, respectively, four activities were studied: (1) a Mg2+-ATPase; (2) a Mg2+-independent activity requiring Ca2+ ions at high concentrations; (3) a (Ca2+ + Mg2+)-ATPase with a high affinity for Ca2+, which were enhanced further (4) by the inclusion of calmodulin (33 nM for half-maximal activity). In the presence of 0.5 mM-EGTA in the buffer used, half saturation for these respective metal ions was observed at 0.9 mM for (1), 1.0 mM for (2), and approximately 0.3 mM for (3) and (4); the latter values correspond to concentrations of free Ca2+ of 0.38 and 0.18 microM for (3) and (4), respectively. The level of activities observed, all in nmol X min-1 X mg-1, under optimal conditions of 37 degrees C, was in a number of preparations (n in parenthesis): for (1) 446 +/- 19 (19); for (2) 362 +/- 18 (3) for (3) 87 +/- 13 (12); and for (4) 161 +/- 29 (12). The (Ca2+ + Mg2+)-ATPase, both in the presence and absence of calmodulin, could be inhibited specifically by a number of agents (approximate I0.5 in parentheses) which, at these concentrations, showed little or no potency against the other activities; among them were vanadate (less than or equal to 10 microM), La3+ (75 microM), trifluoperazine, and other phenothiazines (50 microM). These properties suggest that the (Ca2+ + Mg2+)-ATPase described may be responsible for calcium transport across one (or more) of the several membranes present in nerve endings and contained in the preparation used.
104 citations
TL;DR: The vanadium coordination is tetrahedral and the 17O shifts are correlated closely with formal π-bond order as discussed by the authors, and vanadate is the dominant nucleophile in the two main ring-breaking reactions.
Abstract: Approximately 15 vanadate(V) and polyvanadate(V) species have been identified by 51V n.m.r. in the range pH 7–14, mostly for the first time. Oxygen-17 n.m.r. spectroscopy confirms in several cases that the vanadium coordination is tetrahedral, and that the 17O shifts are correlated closely with formal π-bond order. Most linear and cyclic catenations of VO4 tetrahedra are found up to V6, but there is no branched or triprotonated species or cyclic trimer. Several equilibrium constants and pKa values are reliably measurable. The ion [HVO4]2– rather than OH– is shown to be the dominant nucleophile in the two main ring-breaking reactions, and a simple kinetic pathway is proposed for the formation of decavanadate ion.
104 citations
TL;DR: The results demonstrate that the (K(+),Mg(2+))-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.
Abstract: The (K+,Mg2+)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co2+ > Mg2+ > Mn2+ > Zn2+ > Ca2+) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg2+, the enzyme was further activated by monovalent cations (K+, NH4+, Rb+ ≫ Na+, Cs+, Li+). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K+ approached simple Michaelis-Menten kinetics, with a Km of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K+, and preferred Mn2+ to Mg2+. The results demonstrate that the (K+,Mg2+)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.
93 citations
TL;DR: 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.
TL;DR: Two Ca2+-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca1+ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.
TL;DR: Evidence for this mechanism was obtained by studying vanadate and [48V]vanadate influx into living blood cells (vanadocytes) and the electron paramagnetic resonance (EPR) signal of intracellular vanadyl increases, indicating that transported V(V) is reduced upon entering the cell.
TL;DR: The results show that, like Pi, vanadate accelerates the turnover rate of cross-bridges, but is effective at about 1/500 the concentration required for the Pi effect.
TL;DR: The electrogenic proton-translocating ATPase in the plasma membrane of Neurospora has been solubilized with lysolecithin and purified using a combination of gel filtration and density gradient centrifugation.
TL;DR: In vitro evidence is provided that the Mg(2+)/KCl-ATPase of higher plants is an electrogenic pump, consistent with the hypothesis that an electrogensic H(+) pump is catalyzed by the plasma membrane ATPase of plants.
Abstract: The function of the Mg2+-requiring KCl-stimulated ATPase (ATP phosphohydrolase, EC 3.6.1.3) of higher plants in active ion transport was investigated by using a purified microsomal fraction containing sealed plasma membrane vesicles. (Sze, H. (1980) Proc. Natl. Acad. Sci. USA 77, 5904-5908). A transmembrane electrical potential (+30 to +44 mV), monitored by uptake of a permeant anion (35SCN-), was generated specifically by ATP in purified microsomal vesicles of tobacco callus. ATP-dependent 35SCN- uptake required Mg2+, was optimal at pH 6.75, and showed similar ATP concentration dependence as the Mg2+-requiring KCl-stimulated ATPase activity. Plasma membrane ATPase inhibitors (N,N′-dicyclohexylcarbodiimide and vanadate) inhibited generation of the ATP-dependent electrical potential. A proton conductor (carbonyl cyanide m-chlorophenylhydrazone), but not a K+ ionophore (valinomycin), completely collapsed the electrical potential. The results provide in vitro evidence that the Mg2+/KCl-ATPase of higher plants is an electrogenic pump. These results are consistent with the hypothesis that an electrogenic H+ pump is catalyzed by the plasma membrane ATPase of plants.
TL;DR: Vanadate (10 mM) strongly inhibited endogenous protein degradation as well as the degradation of an exogenous, endocytosed protein (asialofetuin) in isolated rat hepatocytes, suggesting a direct effect upon the lysosomal proteinases.
TL;DR: A survey has been made of the descriptive chemistry of vanadium as it pertains to physiological environments, taking into account the vanadium concentration, pH, coordinating ligands and chelates, presence of other cations, oxidation-reduction potentials and the kinetics of the various vanadium-containing species.
Abstract: A survey has been made of the descriptive chemistry of vanadium as it pertains to physiological environments. Taking into account the vanadium concentration, pH, coordinating ligands and chelates, presence of other cations, oxidation-reduction potentials and the kinetics of the various vanadium-containing species, the following suggestions are made. Free vanadium ions will be monomeric. Monomeric vanadium(V) and vanadium(IV) will each exist in a specific hydrated form. Extracellular vanadium will be in the vanadate, Vv, form. Intracellular vanadium will most likely be predominantly in the vanadyl, Viv, form. Both extracellular vanadium(V) and intracellular vanadium(IV) will be bound to bi- or tridentate ligands if at all. If oxidation-reduction processes involving vanadium are fast relative to transmembrane transport, the transmembrane potential will not be coupled to the Vv/Viv Nernstian potential.
TL;DR: The stimulation of 45Ca-washout presumably reflects an increase in the cytoplasmic Ca2+ level, brought about by an inhibitory effect of vanadate on theCa2+-sensitive ATPase of the sarcoplasmic or the endoplasmsic reticulum.
TL;DR: Evidence is presented that every α subunit can be phosphorylated simultaneously, which rules out the operation of a so-called ‘half-of-the-sites mechanism’, whereby only one of the two α subunits can beosphorylated at any one time.
Abstract: (Na+ + K+)ATPase contains two different subunits, a catalytic subunit (α) and a subunit with uncertain function (β) The enzyme binds ATP, ouabain and vanadate, and can be phos-phorylated by ATP as well as by inorganic phosphate From the previously reported maximal binding and phosphorylation capacities of 35–43 nmol P per mg protein1–3 (based on Lowry protein determination) and the earlier molecular weight value of ∼250,0001,2, a molar binding and phosphorylation capacity of 087–107 mol per mol enzyme was derived As it is generally agreed that the enzyme molecule contains two α subunits1–6 or even a multiple of two7,8, it has been suggested that the enzyme operates by means of a so-called ‘half-of-the-sites mechanism’, whereby only one of the two α subunits can be phosphorylated at any one time9,10 We now present evidence that every α subunit can be phosphorylated simultaneously, which rules out the operation of such a mechanism
TL;DR: Discrete voltage fluctuations that occur spontaneously or in response to dim lights can be recorded from the ventral photoreceptors of Limulus, and these fluctuations are similar to those induced by light.
Abstract: Discrete voltage fluctuations that occur spontaneously or in response to dim lights can be recorded from the ventral photoreceptors of Limulus. The injection of vanadate or the hydrolysis-resistant analog of guanosine triphosphate, GTP-gamma-S, into ventral photoreceptors induces the production of discrete waves in the dark. The chemically induced discrete waves are similar to those induced by light. Ventral photoreceptors may contain a guanyl nucleotide binding protein whose activation by vanadate or GTP-gamma-S induces the discrete waves.
TL;DR: Vanadate (pentavalent vanadium) inhibits with high affinity the ATP-dependent Ca2+ efflux in reconstituted ghosts from human red cell and is potentiated by intracellular Mg2+ and by intrACEllular K+.
TL;DR: The results indicate that the yeast plasma-membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal plasma membranes.
Abstract: A purified preparation of the plasma-membrane ATPase from Saccharomyces cerevisiae was phosphorylated when incubated with (γ-32P]ATP. The phosphoprotein formed has the characteristics of an enzyme intermediate because of its rapidity of phosphorylation and dephosphorylation. When the phosphorylated enzyme was analyzed by polyacrylamide gel electrophoresis in sodium dodecylsulfate only one band with a molecular weight of 100000 contained radioactivity. This band represented about 80% of the protein of the preparation and its enrichment in the course of the purification correlated with the increase in the specific ATPase activity. Both the ATPase reaction and the phosphorylation of the enzyme exhibited an apparent dissociation constant for the enzyme-ATP complex of 0.2 mM, further implicating the phosphoenzyme as an intermediate of the reaction. The sensitivity of the phosphoenzyme bond to alkaline pH and hydroxylamine indicate that it is an acylphosphate. From the maximum level of intermediate (0.7 nmol/mg) and the maximum ATPase activity at 30° C (21 μmol × min−1× mg−1) a turnover number of 30000 min−1 can be calculated. The level of phosphoenzyme was not affected by either the ATPase inhibitors vanadate and dicyclohexylcarbodiimide or by ADP. These results indicate that the yeast plasma-membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal plasma membranes.
TL;DR: A good correlation between the ability of different drugs, known to inhibit ATPase activity, to displace [3H]‐ouabain binding to adreno—medal medullary plasma membranes and their capacity to evoke a CA secretory response from isolated chromaffin cells is demonstrated.
Abstract: 1 Secretion of catecholamines (CA) evoked by ouabain, chlormadinone acetate (CMA), phenoxybenzamine (Pbz) and vanadate, four agents known to inhibit Na(+), K(+)-dependent Mg(2+)-activated adenosine triphosphatase (ATPase) activity has been studied in suspensions of bovine isolated adrenal medullary cells2 Acetylcholine (ACh) evoked a 5 fold increase of the basal CA secretion from isolated cells suspended in oxygenated Krebs-bicarbonate solution kept at 27 degrees C Secretion was antagonized by Ca(2+)-deprivation or hexamethonium, indicating good functional viability of the cells3 Ouabain (10(-7) to 10(-4) M) evoked a progressive, dose-dependent release of CA from cell suspensions Study of the time course of the secretory response for 2 h allowed the separation of two components in the secretory response at all doses studied: a slow initial component (0011 pg/min CA) and a second faster component (0032 pg/min CA)4 CMA evoked a clear-cut CA secretory response The ED(50) for CMA was 10(-4) M, as compared to 3 x 10(-6) M for ouabain Pbz and vanadate did not induce CA release5 [(3)H]-ouabain was taken up and bound to intact isolated cells by a non-saturable binding process However, in semi-purified plasma membranes from bovine adrenal medulla a saturable specific [(3)H]-ouabain binding process was observed with a K(D) of 81 nM Binding to the membranes was ATP-dependent and antagonized by K(+)6 [(3)H]-ouabain specific binding to membranes was antagonized by ouabain and CMA, but not by Pbz or vanadate; the ID(50) for ouabain and CMA were 10(-6) and 10(-5) M respectively7 Ouabain partially inhibited, in a dose-dependent manner, Na(+), K(+)-Mg(2+) ATPase activity of the semi-purified plasma membranes8 The results demonstrate a good correlation between the ability of different drugs, known to inhibit ATPase activity, to displace [(3)H]-ouabain binding to adreno-medullary plasma membranes and their capacity to evoke a CA secretory response from isolated chromaffin cells The data also suggest that the CA secretory effects of ouabain may not be due simply to inhibition of the Na(+) pump and the subsequent ionic redistribution across the plasma membrane; a second mechanism may also be involved
TL;DR: In this paper, the authors investigated the effect of two non-mitochondrial membrane-bound ATPase inhibitors, diethylstilbestrol (DES) and vanadate, on the transport of H + and K + in maize root segments.
TL;DR: The effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of M g2+ and vanadates for the two enzymes are of the same order of magnitude.
TL;DR: TheVanadate-induced inhibition of 30S dynein Mg-ATPase was noncompetitive in the entire range of ATP concentration examined, and the dissociation constant of vanadate and the molecular weight per enzymatic active site according to the kinetics of tight-binding inhibition with several assumptions was estimated.
Abstract: Inhibitory action of vanadate (orthovanadate and metavanadate) on ciliary dynein adenosinetriphosphatase (ATPase) from Tetrahymena was investigated. The apparent concentrations of vanadate giving half-maximal inhibition of Mg-ATPase activity of various dynein fractions were as follows: the axoneme-bound form of dynein at 100 nM, solubilized crude dynein at 50 nM, 14S dynein at 5 microM, and 30S dynein at 20 nM. The Ca-ATPase of 30S dynein was more than 30-fold less sensitive than its Mg-ATPase, and still less sensitive was the Ca-ATPase of 14S dynein. The Mg-ATPase of 30S dynein was most sensitive to vanadate at neutral pH, and the addition of KCl or NaCl into the assay mixture reduced its sensitivity. Varying the assay temperature between 0 and 37 degrees C affected the sensitivity to a slight extent. Metavanadate was as much a potent inhibitor of dynein ATPase as orthovanadate, but vanadium pentoxide was less potent. When the dynein ATPase activity was reciprocally plotted against the concentration of vanadate (the Dixon plot), the inhibition was proved to be biphasic. At lower concentrations of vanadate, the inhibition was more significant. Therefore the Dixon plot had a downward bent. Reexamination of the Lineweaver-Burk plot of 30S dynein Mg-ATP showed a downward bent, which indicates that 30S dynein may have at least two Km values, ca. 1 microM and 3 microM; or otherwise, 30S dynein might possibly have a negatively cooperative nature (Hill coefficient 0.67). The vanadate-induced inhibition of 30S dynein Mg-ATPase was noncompetitive in the entire range of ATP concentration examined. Since the vanadate-induced inhibition of 30S dynein Mg-ATPase could be classified into "tight-binding inhibition", we could estimate the dissociation constant of vanadate and the molecular weight per enzymatic active site according to the kinetics of tight-binding inhibition with several assumptions. Thus, the dissociation constant was 10-15 nM, depending on the ATPase assay condition, while the molecular weight per enzymatic active site was 420 000-480 000, independent of the assay condition with the assumption that the present 30S dynein preparation is totally pure. This value would be reduced about 20% when the purity was taken into consideration.
TL;DR: Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate.
TL;DR: Inhibition of intestinal muscle NaK-ATPase cannot account for the alterations in contractility, and the excitatory response appeared to be due to the action of vanadate at an intracellular site, possibly by inhibition of a Ca- ATPase that controls intrACEllular Ca2+ levels.
Abstract: Intestinal muscle was more sensitive than vascular muscle to vanadate. Vanadate caused a biphasic response in intestinal muscle; inhibition of spontaneous contractile activity was followed by a return of phasic contractions and a sustained increase in baseline tension. The inhibitory response appeared to be due to vanadate-induced release of an inhibitory transmitter from intramural nerve endings. The excitatory response appeared to be due to the action of vanadate at an intracellular site, possibly by inhibition of a Ca-ATPase that controls intracellular Ca2+ levels. Vanadate did not alter intracellular Na+ and K+ levels in isolated longitudinal muscle. Thus, inhibition of intestinal muscle NaK-ATPase cannot account for the alterations in contractility.