Kenneth Kustin

Glutathione reduces cytoplasmic vanadate. Mechanism and physiological implications.

The mechanism by which cells reduce cytoplasmic vanadium(V) (vanadate) to vanadium(IV) was investigated using the human red cell as a model system. Vanadate uptake by red cells occurs with a rapid phase involving chemical equilibration across the plasma membrane and a slower phase resulting in a high concentration of bound vanadium(IV). The slow phase was inhibited in glucose-starved cells and restored upon addition of glucose indicating an energy requirement for this process. The time course of vanadium(IV) appearance (monitored by EPR spectroscopy of intact cells) paralleled the slow phase of uptake indicating that this phase involves vanadium reduction. The reduction of intracellular vanadate to vanadium(IV) was nearly quantitative after 23 h. The intracellular reduction is not enzymatic, since a similar time course of vanadium reduction and binding to hemoglobin was observed when glutathione was added to a hemoglobin + vanadate solution in vitro. Vanadium(IV) binding to hemoglobin was reduced by addition of ATP, 2,3-diphosphoglycerate or EDTA, probably through chelation of the cation. The stability constant of the ATP-vanadium (IV) complex was determined to be 150 M-1 at pH 4.9. The time course of red cell vanadate uptake and reduction was followed in the concentration range in which approximately 60% inhibition of the (Na+ + K+)-ATPase is observed. It is concluded that vanadate is reduced by cytoplasmic glutathione in this concentration range and that the reduction explains the resistance of the (Na+ + K+)-ATPase to vanadium in intact cells.

Vanadium: A Biologically Relevant Element

Publisher Summary This chapter focusses on the properties of the novel vanadium-containing bromoperoxidases. Vanadium has been shown to be an essential requirement in the biological systems and the chemistry of the element is gaining considerable interest. As the chemistry of vanadium is of direct relevance to the mechanism of action of bromoperoxidases, the chemistry of vanadate, properties of vanadium (V) complexes, and their reactivity, including reactions with peroxide, are discussed in the chapter. The work in three selected areas, vanadium in mushrooms, vanadium in oil and coal, and vanadium in tunicates, are also discussed in the chapter. Vanadium is also an essential element for some marine macro-algae, such as the brown seaweed F. spiralus and the green seaweed Enteromorpha compressa. Most assay methods to detect bromoperoxidase activity are based on the bromination of monochlorodimedone, a cyclic diketone that has high affinity for HOBr. The steady-state kinetics of the reaction of vanadium bromoperoxidase with hydrogen peroxide and bromide has been extensively studied. Hypobromous acid is known to be in rapid equilibrium with molecular bromine and tribromide ions in aqueous solutions. The first electron paramagnetic resonance (EPR) spectrum reported of an extract of the cap of the mushroom showed clearly an EPR signal characteristic of oxo-vanadium (IV). Tunicates, commonly called “sea squirts,” are very successful marine organisms found in all oceans of the world.

A systematically designed homogeneous oscillating reaction: the arsenite-iodate-chlorite system

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 A systematically designed homogeneous oscillating reaction: the arsenite-iodate-chlorite system Patrick De Kepper, Irving R. Epstein, and Kenneth Kustin J. Am. Chem. Soc., 1981, 103 (8), 2133-2134• DOI: 10.1021/ja00398a061 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.acs.org on April 10, 2009

Accumulation of vanadium by tunicate blood cells occurs via a specific anion transport system

Tunicates, or sea squirts, are known to sequester vanadium to very high concentrations within specialized blood cells. They selectively accumulate the element from seawater against a 106- to 107-fold concentration gradient, and store it mainly as V(III). The mechanism for this selective accumulation involves the facilitated diffusion of vanadate across the blood cell plasma membrane followed by intracellular reduction to a non-transportable cation. Evidence for this mechanism was obtained by studying vanadate and [48V]vanadate influx into living blood cells (vanadocytes). Influx of [48V]vanadate into the cells is a rapid (t12 = 57 s at 0°C) process which can be saturated (Km = 1.4 (±2%) mM). Net vanadate accumulation is equal to isotopic influx, and accumulated vanadate is not released by washing cells with EDTA. Uncouplers of oxidative phosphorylation and glycolytic inhibitors have no effect on the rate of influx. Phosphate competes with vanadate for transport, and is itself taken up by the cell. The similar anions, sulfate and chromate, neither inhibit transport, nor are they taken up by the vanadocyte. Influx is inhibited by those stilbene disulfonate derivatives known to bind specifically to the external transport site of the anion exchange protein in the human erythrocyte membrane. During the influx of vanadate, the electron paramagnetic resonance (EPR) signal of intracellular vanadyl increases, indicating that transported V(V) is reduced upon entering the cell. The EPR signal of the blood cells at room temperature is characteristic of unbound V(IV), in agreement with reports that reduced vanadate is not bound to a protein or other macromolecule in these cells.

The New Biochemistry of Vanadium

Abstract In the short time since 1977, when it was discovered that vanadate is a potent inhibitor of the sodium pump (Na,K-ATPase), vanadium has become a valuable and widely used probe of enzyme function and mechanism. In the V(V) oxidation state, as diamagnetic vanadate, it mimics phosphate and interacts with a large class of phosphatases and phosphotransferases. In the V(IV) oxidation state, as paramagnetic vanadyl, it behaves as a transition metal ion and can replace other such ions in metalloproteins. Because the potentials of V(V) and V(IV) lie within limits tolerated by living systems, vanadium can also participate in many biological electron transfer reactions. The wide variety of effects reported in the literature that arc observed when vanadium is added exogenously to enzymes, cells and tissues can be largely explained by consideration of the principles of aqueous inorganic vanadium chemistry. The true role of endogenous vanadium in living systems remains obscure, however.

The blood of Ascidia nigra: blood cell frequency distribution, morphology, and the distribution and valence of vanadium in living blood cells.

1. The blood plasma of Ascidia nigra has been characterized with regard to pH, salinity, and ultraviolet-visible absorption. Whole blood of A. nigra has been characterized with regard to cell count, cellular volume, hemoglobin (iron) content, vanadium content. Blood cell types have been examined, categorized, and differentially counted.2. Epr and nmr studies prove that trivalent vanadium is present. Staining of whole blood slides with OsO4 reagent identify the vanadium-carrying cells as being mainly the green globular blood cells. The observed uv-vis absorption spectrum does not correlate well with known vanadium(III) complex spectra.3. Density separation of blood cells has been achieved; this result coupled with various cell morphologies suggests that different cells may represent different maturational stages in a developmental process.4. Vanadium analyses of layerings of cells of different densities suggest that vanadium may be present in more than one type of blood cell, where, when not present as van...

Distribution of tunichrome and vanadium in sea squirt blood cells sorted by flow cytometry.

Specialized blood cells of many tunicates accumulate high concentrations of vanadium and phenolic peptide pigments called tunichromes (TC). In order to determine whether V and TC reside in the same cells,Ascidia nigra andAscidia ceratodes blood cell subpopulations were isolated by fluorescence-activated cell sorting (flow cytometry) and chemically analyzed. V was found in the spherical, gree/grey signet ring cells, and to a lesser degree in the mulberry-shaped, yellow/ green morula cells (MRs), whereas free TC was detected mainly in MRs.

Do vanadate polyanions inhibit phosphotransferase enzymes

Decavanadate inhibits hexokinase, adenylate kinase and phosphofructokinase; neither mono-, tri nor tetrameric vanadate anion is an inhibitor. Decavanadate inhibits phosphofructokinase obtained from bacterial and protistic sources. No form of vanadium(V) anion inhibits galacto-, glycero-, pyruvate and creatine kinase, or inorganic pyrophosphatase. Decavanadate appears to be a non-competitive inhibitor of both hexokinase substrates.

Vanadium in tunicates: Oxygen-binding studies

1. 1. No reversible oxygen-binding by the blood of the tunicate A. nigra is detectable. 2. 2. Blood cells contain iron(II) and vanadium(III), and a yellow-green chromogen which can be separated from the metals by fractionation on Sephadex G-100. 3. 3. The chromogen will reduce oxygen in alkaline medium, it is heat-stable and has absorption peaks at 280 and 330 nm (in 0.1 N HCl). The peak at 330 nm is lost on oxidation.

Vanadium-containing tunicate blood cells are not highly acidic

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [14C] methylamine. The pH of unfractionated blood cells is 7.39 +/- 1.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.