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Showing papers on "Vanadate published in 1989"


Journal ArticleDOI
TL;DR: It is found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase, which suggests that CPA interferes with the ATP-induced conformational changes related to Ca2- transport.

748 citations


Journal ArticleDOI
TL;DR: Wehrli and Stumm as mentioned in this paper showed that the rate of oxidation of VO2+ by oxygen is significantly enhanced by hydrolysis or adsorption to hydrous oxide surfaces and showed that coordinated OH-groups of solid surfaces are able to mediate the electron transfer from the metal ions to the O2-molecule.

364 citations


Journal ArticleDOI
TL;DR: Arsenate proved to be competitive with the highand low-affinity phosphate uptake system and induced transient membrane potential changes of up to 120 mV which were similar to those induced by phosphate and indicated a cotransport mechanism with at least 2H+/H2As04.
Abstract: Interference of arsenate and vanadate with phosphate uptake in Lemna gibba L. was studied by measuring voltage changes and (32P)phosphate uptake. Arsenate proved to be competitive with the highand low-affinity phosphate uptake system. It induced transient membrane potential changes of up to 120 mV which were similar to those induced by phosphate and indicated a cotransport mechanism with at least 2H+/H2As04. The amplitude of the transient arsenate-induced membrane depolarization was strongly influenced by phosphate starvation. A permanent membrane depolarization to the diffusion potential was achieved within 2 to 6 h in P-starved plants. Thus, arsenate is indeed a strongly competitive physiological analogue of phosphate in higher plants. Vanadate was easily transported into L. gibba as concluded from transient Em changes of up to 110 mV. Vanadate interfered only slightly and non-specifically with the two phosphate transport mechanisms. Like phosphate, vanadate uptake seems to be an H+-cotransport mechanism, both with similar optima at pH 6 0. Unlike phosphate uptake, vanadate-linked membrane depolarization was not affected by high intracellular phosphate concentrations. P-starvation did not enhance the weak long-term effect on Em. Hence, vanadate, in contrast to arsenate, is not regarded as a physiological phosphate analogue. The distinct and rapid vanadate-induced and permanent membrane depolarization of Avena sativa, Triticum aestivum and Glycine max leaves was not seen in Lemna nor in leaves of Gossypium hirsutum and Nicotiana tabacum. Plasmalemma-enriched preparations of L. gibba revealed, however, a high vanadate-sensitive ATPase activity (87%). As a possible explanation for these differences it is suggested that the latter plant species have cytosolic vanadate-detoxifying properties, i.e. they can reduce vanadate to vanadyl ions, in contrast to the former group of plant species.

319 citations


Journal ArticleDOI
TL;DR: It is reported that pervanadate mimics insulin in isolated rat adipocytes to stimulate lipogenesis, inhibit epinephrine-stimulated lipolysis, and stimulate protein synthesis, which is 10(2)-10(3) times more potent than vanadate alone.
Abstract: Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

304 citations


Journal ArticleDOI
TL;DR: Interactions of vanadate and vanadyl cation with potential ligands including buffers, chelating agents, enzyme substrates, cofactors, amino acids, peptides, and proteins were examined and seven out of 26 commonly used buffers were found to strongly complex vanadates.
Abstract: Enzyme kinetics have been used to study interactions of trace-level concentrations of vanadate (V(V)) and vanadyl cation (V(IV)) in biological systems. A quantitative method based on the inhibition of alkaline or acid phosphatase by monomeric vanadate or vanadyl cation has been developed to determine the concentration of free monomeric vanadate or vanadyl cation at 10{sup {minus}5}-10{sup {minus}7} M vanadium concentrations. Interactions of vanadate and vanadyl cation with potential ligands including buffers, chelating agents, enzyme substrates, cofactors, amino acids, peptides, and proteins were examined. Seven out of 26 commonly used buffers were found to strongly complex vanadate, and an additional 11 buffers were found to complex vanadate to various degrees. The vanadyl cation generally interacts more strongly with these buffers than does vanadate.

166 citations


Journal ArticleDOI
TL;DR: The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H(+)-ATPase activity in vivo.
Abstract: Lysophosphatidylcholine at concentrations of 30 micromolar stimulated the rate of MgATP-dependent H+-accumulation in oat (Avena sativa L. cv Rhiannon) root plasma membrane vesicles about 85% while the passive permeability of H+ was unchanged. Activation was dependent on chain length, degree of saturation, and head group of the lysophospholipid. A H+-ATPase assay was developed that allowed the simultaneous measurement of proton pumping and ATPase activity in the same sample. ATP hydrolysis was also stimulated by lysophospholipids and showed the same lipid specificity, but stimulation was only about 25% at 30 micromolar. At higher concentrations of lysophosphatidylcholine the ATPase activity in a latency-free system could be stimulated about 150%. The enzymic properties of proton pumping and ATP hydrolysis were otherwise identical with respect to vanadate sensitivity, Km for ATP and pH optimum. The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H+-ATPase activity in vivo.

147 citations


Journal ArticleDOI
TL;DR: The results suggest that the defect in muscle glycogen synthesis is the major determinant of insulin resistance in diabetic rats and both vanadate and phlorizin treatment normalize meal tolerance and insulin sensitivity in diabetic Rats.
Abstract: Vanadate has insulin-like activity in vitro and in vivo. To characterize the in vivo mechanism of action of vanadate, we examined meal tolerance, insulin-mediated glucose disposal, in vivo liver and muscle glycogen synthesis, and in vitro glycogen synthase activity in 90% partially pancreatectomized rats. Four groups were studied: group I, sham-operated controls; group II, diabetic rats; group III, diabetic rats treated with vanadate; and group IV, diabetic rats treated with phlorizin. Insulin sensitivity, assessed with the euglycemic hyperinsulinemic clamp technique in awake, unstressed rats, was reduced by approximately 28% in diabetic rats. Both vanadate and phlorizin treatment completely normalized meal tolerance and insulin-mediated glucose disposal. Muscle glycogen synthesis was reduced by approximately 80% in diabetic rats (P less than 0.01) and was completely restored to normal by vanadate, but not by phlorizin treatment. Glycogen synthase activity was reduced in skeletal muscle of diabetic rats (P less than 0.05) compared with controls and was increased to supranormal levels by vanadate treatment (P less than 0.01). Phlorizin therapy did not reverse the defect in muscle glycogen synthase. These results suggest that (a) the defect in muscle glycogen synthesis is the major determinant of insulin resistance in diabetic rats; (b) both vanadate and phlorizin treatment normalize meal tolerance and insulin sensitivity in diabetic rats; (c) vanadate treatment specifically reverses the defect in muscle glycogen synthesis in diabetic rats. This effect cannot be attributed to the correction of hyperglycemia because phlorizin therapy had no direct influence on the glycogenic pathway.

126 citations


Journal ArticleDOI
TL;DR: The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the Delta mu H+ it generates.

122 citations


Journal ArticleDOI
TL;DR: Ca2+ uptake and Ca2+ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells are measured.
Abstract: We have measured Ca2+ uptake and Ca2+ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca2+ uptake into cells was monitored with a Ca2+ electrode, whereas Ca2+ uptake into membrane vesicles was measured with 45Ca2+. Using inhibitors of known action, such as the H+ ATPase inhibitors NBD-Cl and NEM, the Ca2+ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP3) and its analog inositol 1,4,5-trisphosphorothioate (IPS3), we could functionally differentiate two nonmitochondrial Ca2+ pools. Ca2+ uptake into the IP3-sensitive Ca2+ pool (IsCaP) occurs by a MgATP-dependent Ca2+ uptake mechanism that exchanges Ca2+ for H+ ions. In the absence of ATP Ca2+ uptake can occur to some extent at the expense of an H+ gradient that is established by a vacuolar-type MgATP-dependent H+ pump present in the same organelle. The other Ca2+ pool takes up Ca2+ by a vanadate-sensitive Ca2+ ATPase and is insensitive to IP3 (IisCaP). The IsCaP is filled at "higher" Ca2+ concentrations (approximately 10(-6) mol/liter) which may occur during stimulation. The low steady-state [Ca2+] of approximately 10(-7) mol/liter is adjusted by the IisCaP. It is speculated that both Ca2+ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.

116 citations


Journal ArticleDOI
TL;DR: Vanadate's insulin-mimetic effect on mouse diaphragm glycogenesis occurs at a site distal to the insulin receptor, suggesting that vanadate’s insulin-receptor activation by in vivo Vanadate administration is based on a conformational change in the receptor beta-subunit upon autophosphorylation.
Abstract: The in vivo administration of sodium orthovanadate stimulated the incorporation of [14C]glucose into [14C] glycogen, in a dose- and time-dependent manner, in mouse diaphragm. Activation of diaphragm insulin receptor was measured by exogenous tyrosine kinase activity and an antibody that recognizes a conformational change in the receptor β-subunit upon autophosphorylation. Neither method detected insulin receptor activation by in vivo vanadate administration, suggesting that vanadate's insulin-mimetic effect on mouse diaphragm glycogenesis occurs at a site distal to the insulin receptor. (Endocrinology 124: 1918-1924, 1989)

113 citations


Journal ArticleDOI
TL;DR: These results further demonstrate the usefulness of hygromycin B as a generalized screening tool for isolating diverse plasma membrane ATPase mutants and suggest that Ser-368 and Pro-640 influence the affinity of the phosphate-binding site for Pi.

Journal ArticleDOI
TL;DR: Vanadate exerts an insulin-like effect on lipolysis, yet its effects on tyrosine phosphorylation differ from those of insulin.
Abstract: Tyrosine phosphorylation of the insulin receptor and other intracellular proteins in rat adipocytes was examined using an immunoblot technique with antiphosphotyrosine antibody. Insulin at 10-7 M increased the tyrosine phosphorylation of the 95K subunit of the insulin receptor (15-fold) and proteins of 180K (7-fold) and 60K (23-fold). Increases in insulin-dependent phosphorylation of the three proteins were detectable at 1O-10 M insulin and attained steady state within 30 sec of insulin (10-7 M) addition. Small effects of insulin (<30% increases) were observed on proteins of 120K and 53K. In contrast to insulin, the effects of vanadate on tyrosine phosphorylation were small and nonspecific. Vanadate increased tyrosine phosphorylation of the 95K insulin receptor /3-subunit and the 120K and 60K proteins similarly, with increases of 1.5- to 3-fold at 1 mM and 2-fold or less at 200 and 50 μM. Vanadate-dependent tyrosine phosphorylation of the 180K protein increased to a maximum of only 30% at 200 μM. Tyrosine...

Journal ArticleDOI
TL;DR: It is suggested that the structural differences seen in the crystal structures of free and complexed ribonuclease T1 are related to conformational adjustments associated with the substrate binding process.
Abstract: Ribonuclease T1 was crystallized in the presence of vanadate(V). The crystal structure was solved by molecular replacement and refined by least-squares methods using stereochemical restraints. The refinement was based on data between 10 and 1.8 A and converged at a crystallographic R factor of 0.137. Except for the substrate-recognition site the three-dimensional structure of ribonuclease T1 closely resembles the structure of the enzyme complexed with guanosine 2'-phosphate and its derivatives. A tetrahedral anion was found at the catalytic site and identified as H2VO4-. This is the first crystal structure of ribonuclease T1 determined in the absence of bound substrate analogue. Distinct structural differences between guanosine-free and complexed ribonuclease T1 are observed at the base-recognition site: The side chains of Tyr45 and Glu46 and the region around Asn98 changed their conformations, and the peptide bond between Asn43 and Asn44 has turned around by 140 degrees. We suggest that the structural differences seen in the crystal structures of free and complexed ribonuclease T1 are related to conformational adjustments associated with the substrate binding process.

Journal ArticleDOI
TL;DR: In this paper, the authors used 51V NMR spectra of vanadate addition to Fe,Tf and Fec-Tf to characterize the metal binding sites of transferrin.
Abstract: 5'V NMR is a highly sensitive tool to probe the nature of the metal binding sites of human transferrin (Tf). At a field strength of 11.7 T, the two vanadium(V) binding sites in V,-Tf are characterized by 51V chemical shifts at -529.5 and -53 1.5 ppm versus VOCl,. These shifts are assigned to the Cand N-terminal sites, respectively, based on the 51V NMR spectra of vanadate addition to FeN-Tf and Fec-Tf. Tight binding of V(V) to the metal binding site is further ascertained by a linear increase of signal area with V(V) concentration up to an approximate 2:l stoichiometry, stoichiometric displacement of protein-bound V(V) during titration with Fe(III), and the absence of the -529.5/-531.5 ppm resonance upon addition of V(V) to Fe,Tf and Ga2Tf. The chemical shift of the Vc-Tf resonance is independent of pH (5.8-9.0) and temperature (275-310 K), whereas the VN-Tf resonance varies slightly (1-2 ppm) with pH and temperature. At relatively high V(V) concentrations and at high ratios of V(V)/Tf, conditions under which a large fraction of the V(V) is present as V oligomers, the 51V resonance of Tf-bound V(V) is not observed, possibly owing to interference by the oligomeric species. Under conditions of tight V(V) binding, the sharpness of the protein-bound resonances is a consequence of the motional characteristics of transferrin, which place the Tf-bound vanadium(V) outside the extreme narrowing limit but within the motional narrowing limit. Several theoretically predicted consequences for an I = 7/2 nucleus are observed experimentally, some for the first time in an aqueous protein system. (1) The observed signal intensity of transferrin-bound vanadium(V) compared to that of an equimolar aqueous vanadate sample is substantially reduced, since only one out of four spinspin relaxation components (Le., + I / * I / * transition) is observed. (2) At 7.05 T the line width is substantially greater than at 11.7 T, and a 5-ppm upfield shift is observed which is attributed to a dynamic frequency shift. (3) The line width of Tf-bound V(V) is not affected by solvent viscosity up to 50% v/v glycerol/buffer. (4) Measurements of signal intensity as a function of pulse length reveal increased 51V precession frequencies in the radio-frequency field, as frequently observed for studies of half-integer quadrupolar nuclei in the solid state. The excitation spectrum is found to be identical for Vc and VN sites and independent of the V/Tf ratio. At small pulse angles, the fraction of the signal observed is constant and amounts to ca. 20% of that of an equimolar solution of free vanadate, in close agreement with the theoretical prediction. The present study confirms that 51V NMR is a powerful tool to probe V(V) binding to apotransferrin and should also become widely applicable to characterize V(V) binding to other macromolecules. Transferrins are glycoproteins whose primary function is to bind and transport iron. Transferrins also coordinate a wide variety of other metal ions (e.g., Cu(II), V02+, Cr(III), Ga(III), TI(III), etc.),' including v a n a d i ~ m ( V ) . ~ ~ Human transferrin (Tf) is a single polypeptide chain with two homologous regions each of which binds one metal atom.' Elucidation of the similarities and differences between the two metal binding sites continues to be an area of active interest, the results of which should increase our understanding of the functional differences of the two binding sites. The X-ray crystal structure of human lactoferrin shows that the iron binding sites are separated by 42 8, and that the metal binding ( l ? (a) Chasteen, N. D. Adu. Inorg. Biochem. 1983, 58 201. (b) Aisen, P.; Listowsky, I . Annu. Reo. Biochem. 1980, 49, 357. (c) Brock, J. H. in Metalloproteins; Harrison, P. M., Ed.; Verlag Chemie: Weinheim, West Germany, 1985; Part 2, p 183 and references therein. (2) Harris, W. R.; Carrano, C. J . J . Inorg. Biochem. 1984, 22, 201. (3) Chasteen, N. D.; Grady, J. K.; Holloway, C. E. Inorg. Chem. 1986, (4) Butler, A.; Danzitz, M. J.; Eckert, H. J . Am. Chem. Sot. 1987, 1/39, 25, 2754. 1864. ligands at both binding sites are two tyrosine residues, one histidine residue, one aspartate residue, a H,O (or OH-) molecule, and an anion (COj2or HCO;).5 The fact that human transferrin and lactoferrin share a high degree of sequence homology (30%),Ic that all the metal binding amino acids in lactoferrin are conserved in human transferrin, and that much spectroscopic evidence indicates that the binding sites of lactoferrin and human transferrin are similar, suggests that the metal binding ligands are the same between transferrin and lactoferrin and between the two metal binding sites in human transferrin. A wide variety of experimental results, however, suggest the two metal binding sites in human transferrin are not equiualent, even though the ligands of both sites may be identical. Structural differences have been inferred from the ESR spectra of the Fe(HI)-: V02+-,7 Cu(11)-, and Cr(III)-bound8 transferrin derivatives ( 5 ) Anderson, B. F.; Baker, H. M.; Dodson, E. J.; Norris, G. E.; Rumball, S. V.; Waters, J. M.; Baker, E. D. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, (6) Aisen, P.; Liebman, A.; Zweier, J. J . Eiol. Chem. 1978, 253, 1930. 1769-1773. 0002-7863/89/1511-2802$01.50/0

Journal ArticleDOI
TL;DR: Vanadate treatment was found to have a normalizing effect on blood glucose levels in diabetic animals as well as restoring amylase content in the pancreas of diabetic animals.
Abstract: In the present study, streptozotocin-induced diabetic rats with their corresponding controls, were treated orally with sodium metavanadate A gradual increase of the vanadate concentration up to 08 mg/ml in the drinking water, lowered the blood glucose levels of the diabetic animals to normal values without changing the insulin levels On the other hand, vanadate did not affect the blood glucose levels of the non-diabetic animals; it did however induce lower levels of circulating insulin in these animals The lowering of the glycaemic values of the diabetic animals was closely related to the consumption of vanadate When the treatment was ceased, the blood glucose levels rose rapidly The diabetic animals responded to the vanadate treatment with two sensitivities; while the large majority of the diabetic animals displayed stable normoglycaemic values, others had fluctuating values Amylase content in the exocrine pancreas of these two subgroups of animals was studied separately and compared to that from the non-treated control and diabetic animals The presence of amylase in the pancreatic acinar cells was assessed by the protein A-gold immunocytochemical approach and biochemical determinations Amylase was found to be very low in the non-treated diabetic animals Lowering of the blood glucose levels induced by the vanadate treatment restored the amylase to levels similar to those of the controls However, vanadate-treated diabetic animals with fluctuating levels of blood glucose, demonstrated only a partial recovery of amylase Thus, vanadate treatment was found to have a normalizing effect on blood glucose levels in diabetic animals as well as restoring amylase content in the pancreas of diabetic animals This appeared to be closely related to the glycaemic values of the diabetic animals

Journal ArticleDOI
TL;DR: The addition of vanadate and molybdate to electropermeabilized platelets induced an increase in serotonin and PDGF secretion and the secretion of these components tightly correlated in a time- and dose-dependent fashion with the phosphorylation of the 50-kDa protein on tyrosyl residues, suggesting that the tyrosine phosphorylated protein may be closely linked to the platelet activation cascade.
Abstract: Addition of vanadate and molybdate to electropermeabilized human platelets caused a time- and dose-dependent increase in the phosphotyrosyl content of 50- and 38-kDa proteins. This effect can most likely be attributed to an inhibition of protein-tyrosine-phosphatase activity because vanadate and molybdate inhibited this activity in platelet extracts by greater than 97% while causing an increase in tyrosyl phosphorylation of artificial substrates that had been added to the same extracts. The addition of vanadate and molybdate to the electropermeabilized platelets also induced an increase in serotonin and PDGF secretion. Interestingly, the secretion of these components tightly correlated in a time- and dose-dependent fashion with the phosphorylation of the 50-kDa protein on tyrosyl residues. This suggests that the tyrosine phosphorylation of this protein may be closely linked to the platelet activation cascade.

Journal ArticleDOI
TL;DR: The results demonstrate the presence of a Ca2+-ATPase in the plasma membrane of C. communis and makes it a likely component involved in adjusting low cytoplasmic Ca2+, as well as highlighting the nucleotide specificity and the sensitivity towards vanadate.
Abstract: Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U3+U3′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven 45Ca2+ transport [? 15 pkat (mg protein)−1]. Ca2+ uptake into the vesicles had a pH optimum of 7.2 and apparent Km values for Ca2+ of 4.4 μM and for Mg-ATP of 300 μM. Ca2+ uptake, K+, Mg2+-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm−3) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca2+ transport into the vesicles, excluding a Ca2+/H+ exchange driven by a proton gradient. ATP-dependent Ca2+ uptake was inhibited by erythrosin B (I50= 0.1 μM), ruthenium red (I50= 30 μM), La3+ (I50= 10 μM) and vanadate (I50= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I50= 70 μM) and W-7 (I50= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca2+ efflux from the vesicles rather than inhibit Ca2+ uptake. Our results demonstrate the presence of a Ca2+-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca2+ out of the cell. Its high affinity for Ca2+ makes it a likely component involved in adjusting low cytoplasmic Ca2+ levels. No indications for a secondary active Ca2+/H+ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca2+-ATPase from the H+-translocating K+. Mg2+-ATPase in C. communis plasma membranes.

Journal ArticleDOI
TL;DR: The detailed purification of this mitogen-activated S6 kinase from high-speed supernatants of vanadate-treated cell extracts is described, and the specific activity of the homogeneously purified enzyme is 0.6 mumol/min/mg of protein.

Journal ArticleDOI
TL;DR: In this article, the equilibrium concentrations of Na3VO4, NaVO3, and V2O5 in a mixed sodium sulfate-vanadate solution containing 30 mol% vanadate as a function of melt basic were derived.
Abstract: Thermodynamic calculations for the equilibrium concentrations of Na3VO4, NaVO3, and V2O5 in a mixed sodium sulfate-vanadate solution containing 30 mol% vanadate as a function of melt basic...

Journal ArticleDOI
TL;DR: Results are consistent with a CO(2) concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.
Abstract: Neither Dunaliella cells grown with 5% CO2 nor their isolated chloroplasts had a CO2 concentrating mechanism. These cells primarily utilized CO2 from the medium because the K(0.5) (HCO3−) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K(0.5) CO2 was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO2 concentrating mechanism was present that decreased the K0.5 (CO2) to about 0.5 micromolar and K0.5 (HCO3−) to 11 micromolar at pH 8. These K0.5 values suggest that air-adapted cells preferentially concentrated CO2 but could also use HCO3− from the medium. Chloroplasts isolated from air-adapted cells had a K(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO2. Chloroplasts from air-adapted cells, but not CO2-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO2 concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO2 concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO2 concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.

Journal ArticleDOI
TL;DR: The results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na, k-ATpase and the gastric H, K- ATPase enzymes.

Journal ArticleDOI
TL;DR: A 72 kDa protein was phosphorylated when membranes were incubated with Mg 2+ and [ 32 P]orthophosphate, and phosphorylation was inhibited by both vanadate and delta endotoxin, suggesting that destabilization of this phosphoprotein was responsible for phosphatase inhibition.

Journal ArticleDOI
TL;DR: The activation of electrogenic H+ extrusion and of K+ uptake by light is mediated by some products of photosynthesis, and the identification of this system with the plasma membrane H+-ATPase is indicated by the observed inhibition of the effects of either light or fusicoccin by the H + - ATPase inhibitors vanadate and erythrosin B.
Abstract: In Elodea densa leaves light strongly stimulates electrogenic, K+-dependent, vanadateand erythrosin B-sensitive H+ extrusion and hyperpolarizes the transmembrane electrical potential. These effects of light are suppressed by treatment with DCMU, an inhibitor of photosynthesis, which has no effect on H + extrusion in the dark. Light-induced H + extrusion requires the presence of K+ in the medium and is associated with increased K+ uptake and alkalinization of the cell sap. Light-induced H+ extrusion increases with increased C02 concentration. At constant C02 concentration (104 parts 10 ~6) the rate of H+ extrusion is strongly enhanced by an increased light intensity up to 30 W m 2. Different wavelengths, between 400 and 730 nm, induce a significant stimulation of both proton secretion and transmembrane potential hyperpolarization. The stimulating effects of light on H+ extrusion, K+ uptake and cell sap pH are very similar to those induced in the dark by fusicoccin, a toxin known to stimulate strongly ATP-driven, vanadateand erythrosin B-sensitive H+ transport. In the light, the effects of fusicoccin are only partially additive to those of light, thus suggesting that the two factors influence the same system. The identification of this system with the plasma membrane H+-ATPase is indicated by the observed inhibition of the effects of either light or fusicoccin by the H + -ATPase inhibitors vanadate and erythrosin B. These data indicate that the activation of electrogenic H+ extrusion and of K+ uptake by light is mediated by some products of photosynthesis. The mechanism and the possible physiological implications of this phenomenon are discussed.

Journal ArticleDOI
TL;DR: Findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2- ATPases.


Journal ArticleDOI
TL;DR: It is concluded that cholinergic vesicles contain a P‐ type ATPase of unknown function and a V‐type ATPase which is the proton pump.
Abstract: Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 μM vanadate. This activity is due to a recently purified phos-phointermediate, or P-type, ATPase. Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethyl-maleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, A-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 μM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilo-dalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump

Journal ArticleDOI
TL;DR: The purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 microM) is reported, unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Journal ArticleDOI
TL;DR: The results suggest that theCa2+ release initiated by lithocholate and its conjugates results from a direct action on the Ca2+ permeability of the Ins(1,4,5)P3-sensitive pool.

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TL;DR: The organometallic oxide clusters (MCp*)4V6O19] (M = Rh, Ir; Cp* = C5Me5) were prepared and characterized by elemental analyses, SIMS as well as IR and NMR (1H, 13C, 17O, 51V) spectroscopy as discussed by the authors.
Abstract: The organometallic oxide clusters [(MCp*)4V6O19] (M = Rh, Ir; Cp* = C5Me5) were prepared and characterized by elemental analyses, SIMS as well as IR and NMR (1H, 13C, 17O, 51V) spectroscopy Single crystal X-ray analysis showed that [(RhCp*)4V6O19]·4CH3CN·H2O contains the vanadate hexamer core (V6O19) The rhodium cluster displays site selective oxygen exchange with free water at the bridging oxygen atoms

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TL;DR: P pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.