Showing papers on "Vanadate published in 1984"

Uptake of Metal Ions by Rhizopus arrhizus Biomass

Abstract: Rhizopus arrhizus biomass was found to absorb a variety of different metal cations and anions but did not absorb alkali metal ions. The amount of uptake of the cations was directly related to ionic radii of La3+, Mn2+, Cu2+, Zn2+, Cd2+, Ba2+, Hg2+, Pb2+, UO22+, and Ag+. The uptake of all the cations is consistent with absorption of the metals by sites in the biomass containing phosphate, carboxylate, and other functional groups. The uptake of the molybdate and vanadate anions was strongly pH dependent, and it is proposed that the uptake mechanism involves electrostatic attraction to positively charged functional groups.

Mechanisms of Action of Vanadium

Abstract: Vanadium is widely distributed, the twenty-first most abundant element in the Earth's crust, with an average content of 135 ppm. In sea water, vanadium ranks thirty-fourth in abundance, with an average concentration of only 2 ppb. Because it evolved as an essential element for certain forms of life and also because of its wide industrial use, the biological actions of vanadium are of interest to scientists. Excellent accounts of the history and previous know ledge of vanadium are available. (1-7). The chemistry of vanadium is complex because the metal can exist in oxidation states from -1 to +5 and forms polymers frequently (8). Recently Rubinson (9) reviewed the material concerning the form of biochemically active vanadium. The following generalizations appear justified. At low­ normal concentrations in mammals and birds, any free vanadium will be in hydrated, monomeric form. In the body fluids at pH 4-8, the predominant species will be VOi ( + 5 oxidation state), vanadate (metavanadate). VOi may enter certain cells by an anion transport system and be reduced by glutathione to VOH (+4 oxidation state), vanadyl. By way of speculation, the oxidation­ reduction reactions may be as follows: H+ + VOi + 2GSH � Y02+ + G2S2 + OH+ e + H20. Extensive binding to extraand intracellular ligands may be expected. Since phosphate and Mg2+ are ubiquitous in biological processes, YOi as the analogue of phosphate and Y02+, which resembles the size of Mg2+ (respective ionic radii: 0.60 and 0.65 ft.), potentially have many bio­ chemical and cellular sites of action. For example, vanadium compounds in­ hibit ATP phosphohydrolases, ribonuclease, adenylate kinase, phosphofructo­ kinase, squalene synthetase, glyceraldehyde-3-phosphate dehydrogenase (10),

Effects of Vanadate on the Plasma Membrane ATPase of Red Beet and Corn

Abstract: The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L) and red beets (Beta vulgaris L) The Ki for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar Vanadate inhibition was optimal at pH 65, enhanced by the presence of K+, and was partially reversed by 1 millimolar EDTA The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate Vanadate decreased both the Km and Vmax of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats

Effects of Vanadate on the Plasma Membrane ATPase of Red

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

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts.

Abstract: Recent experiments show that exogenous NADH increases the O2 consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled Vmax of the mechanism I (saturable) component of K+ uptake, while Km was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K+ influx and H+ efflux, while NADH-stimulated K+ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H+ and K+ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K+ influx and a more than 3-fold increase of H+ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K+ uptake.

The Hydrogen Ion-pumping Adenosine Triphosphatase of Platelet Dense Granule Membrane

Abstract: Using a coupled transport assay which detects only those ATPase molecules functionally inserted into the platelet dense granule membrane, we have characterized the inhibitor sensitivity, substrate specificity, and divalent cation requirements of the granule H+ pump. Under identical assay conditions, the granule ATPase was insensitive to concentrations of NaN3, oligomycin, and efrapeptin which almost completely inhibit ATP hydrolysis by mitochondrial membranes. The granule ATPase was inhibited by dicyclohexylcarbodiimide but only at concentrations much higher than those needed to maximally inhibit mitochondrial ATPase. Vanadate (VO,-) ion and ouabain also failed to inhibit granule ATPase activity at concentrations which maximally inhibited purified Na+,K+-ATPase. Two alkylating agents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and N-ethylmaleimide both completely inhibited H+ pumping by the granule ATPase under conditions where ATP hydrolysis by mitochondrial membranes or Na+,K+-ATPase was hardly affected. These results suggest that the H+-pumping ATPase of platelet granule membrane may belong to a class of ion-translocating ATPases distinct from both the phosphoenzymetype ATPases present in plasma membrane and the FIFo-ATPases of energy-transducing membranes.

Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen-stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells.

Abstract: Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H+ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H+ exchange system of A431 cells. Stimulation of Na+/H+ exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promoters also do not modulate the activation of Na+/H+ exchange. By contrast, the stimulation of Na+/H+ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H+ antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H+ exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca2+. The inhibition BY PMA of EGF-promoted Na+/H+ exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H+ exchange by polypeptide growth factors.

A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide.

Abstract: Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized.

Maintained contractions of rat uterine smooth muscle incubated in a Ca2+‐free solution

Abstract: 1 The effects of acetylcholine (10−4m), prostaglandin E2 (10−6m), vanadate (5 × 10−4m) and fluoride (10−2m) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+-free EGTA-containing solutions. 2 All four substances produced maintained contractions which could be initiated repeatedly after exposure to Ca2+-free solution for more than 1 h, without a significant decrease. The largest contractions were obtained with vanadate and the smallest ones with acetylcholine. The tension was usually 7–30% of the control contraction triggered by an action potential in Ca2+ containing solution. 3 Maintained contractions induced by fluoride were unaffected by isoprenaline while those induced by acetylcholine, prostaglandin E2 and vanadate were completely relaxed. 4 Prostaglandin E2 -and vanadate-induced contractions were slightly reduced by Na+ removal or by adding Ca2+ antagonists. In contrast, contractions induced by acetylcholine were suppressed in Na+-free solution and largely inhibited in the presence of Ca2+ antagonists. 5 The depolarization induced by acetylcholine in Ca2+-free solution was strongly dependent on the external Na+ concentration. The relationship between the size of the acetylcholine-induced depolarization and the membrane potential (shifted by constant currents) was linear, giving an apparent reversal potential for acetylcholine close to zero potential. 6 In Ca-free solutions and in the presence of atropine, Na+ action potentials of long duration can be evoked which produced contractions of the same order of magnitude as those initiated by acetylcholine-induced depolarizations. 7 These results are consistent with the hypothesis that the maintained contractions in Ca2+-free solutions induced by several stimulants could be related to Ca2+-independent mechanisms (fluoride) or Ca2+ release from an intracellular store. This latter mechanism would include both pharmacomechanical (prostaglandin E2, vanadate) and electromechanical (acetylcholine) coupling.

Hypothalamic sodium-transport inhibitor is a high-affinity reversible inhibitor of Na+-K+-ATPase

Abstract: Bovine hypothalamus contains a stable, low molecular weight substance with ouabain-like properties. To further study its mechanism of action and potential physiological importance we examined its effects on purified Na+-K+-ATPase in a kinetic coupled-enzyme assay. Under optimal conditions up to 95% of Na+-K+-ATPase activity could be inhibited by the factor. Mg2+ is required for maximal inhibitory activity, but ligand requirements for optimal activity are otherwise distinct from those of both ouabain and vanadate. Inhibition is reversed by high concentrations of sodium chloride plus EDTA. Kinetic analysis yielded a Ki = 1.4 nM. The hypothalamic factor is a high-affinity reversible inhibitor of Na+-K+-ATPase, being at least as potent as the cardiac glycoside ouabain and may be a circulating inhibitor of sodium transport, which appears to be associated with experimental volume-expanded hypertension and human essential hypertension.

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane of Beta vulgaris

Abstract: Characteristics of the native and reconstituted H+-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H+-ATPase retained characteristics similar to those of the native plasma membrane H+-ATPase following reconstitution into proteoliposomes. ATPase activity and H+ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO 3 − was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H+ transport showed a pH optimum of 6.5, required a divalent cation (Co2+>Mg2+>Mn2+>Zn2+>Ca2+), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H+-ATPases were stimulated by monovalent ions. Native plasma membrane H+-ATPase showed an order of cation preference of K+>NH 4 + >Rb+>Na+>Cs+>Li+>choline+. This basic order was unchanged following reconstitution, with K+, NH 4 + , Rb+ and Cs+ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H+ transport, both enzymes required a cation such as K+ for maximal transport activity, but were stimulated preferentially by Cl− even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H+-ATPase.

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Abstract: A vanadate-sensitive H+-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H+-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H+ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H+ transport activity was inhibited half-maximally between 1 and 5 μM vanadate (Na3VO4) and nearly abolished by 100 μM vanadate. ATPase activity of native plasma membrane showed aKi for vanadate inhibition of 9.5 μM, and was inhibited up to 80% by 15 to 20 μM vanadate (Na3VO4). ATPase activity of the reconstituted vesicles showed aKi of 2.6 μM for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.