Kan Kanamori

Vanadium-binding proteins (Vanabins) from a vanadium-rich ascidian Ascidia sydneiensis samea

Since the beginning of the last century, it has been known that ascidians accumulate high levels of a transition metal, vanadium, in their blood cells, although the mechanism for this curious biological function remains unknown. Recently, we identified three vanadium-binding proteins (vanabins), previously denoted as vanadium-associated proteins (VAPs) [Zool. Sci. 14 (1997) 37], from the cytoplasm fraction of vanadium-containing blood cells (vanadocytes) of the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we describe the cloning, expression, and analysis of the metal-binding ability of vanabins. Recombinant proteins of two independent but related vanabins, vanabin1 and vanabin2, bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1x10(-5) and 2.3x10(-5) M, respectively. The binding of vanadium(IV) to these vanabins was inhibited by the addition of copper(II) ions, but not by magnesium(II) or molybdate(VI) ions. Vanabins are the first proteins reported to show specific binding to vanadium ions; this should provide a clue to resolving the problem regarding the selective accumulation of vanadium in ascidians.

Structures and properties of multinuclear vanadium(III) complexes: seeking a clue to understand the role of vanadium(III) in ascidians

Certain ascidians accumulate vanadium(III) to a great extent in their blood cells. The role of vanadium(III) in ascidians is still a controversial issue, though 90 years have already passed since the original finding by Henze was published. In order to obtain further insight into the role of vanadium(III) in ascidian blood cells, the coordination chemistry of vanadium(III) has been developed. This review will survey (1) the vanadium(III) complexes bridged by a single oxo group, focusing on the relationship between the preferential formation of μ-oxo dimers and chemical properties of vanadium(III) complexes; (2) the vanadium(III) complexes containing ligands with a terminal alkoxo functionality; (3) the vanadium(III) complexes with dinucleating ligands, which exhibit diverse bridging modes; (4) the sulfato-containing vanadium(III) complexes with regard to the role of sulfate ions in ascidian blood cells; (5) the multinuclear vanadium(III) complexes with various bridging modes and (6) the magnetic coupling of dinuclear vanadium(III) complexes.

Direct reduction from vanadium(V) to vanadium(IV) by NADPH in the presence of EDTA. A consideration of the reduction and accumulation of vanadium in the ascidian blood cells

Abstract Vanadium(V) species are reduced to vanadium(IV) directly by NADPH in the presence of EDTA. At neutral pH, vanadium(V)–EDTA complex, [VO 2 (EDTA)] 3− (20 mM) was almost completely reduced to [VO(EDTA)] 2− directly by NADPH (200 mM) after 15 h under aerobic and anaerobic conditions. The reduction is markedly accelerated at low pH. At pH 3, the vanadium(V) complex (20 mM) was reduced by NADPH (20 mM) within 1 h. Oxygen had little effect, but inhibited the reduction to some extent at low pH. At low pH, simple vanadium(V) species were also partly reduced without the assistance of EDTA, resulting in the formation of mixed-valence (V(IV)–V(V)) polynuclear species. The accumulation and reduction of vanadium in the vanadocytes of ascidians are discussed based on the present results.

A novel vanadium reductase, Vanabin2, forms a possible cascade involved in electron transfer

The unusual ascidian ability to accumulate high levels of vanadium ions at concentrations of up to 350 mM, a 10(7)-fold increase over that found in seawater, has been attracting interdisciplinary attention for a century. Accumulated V(V) is finally reduced to V(III) via V(IV) in ascidian vanadocytes. Reducing agents must therefore participate in the reduction. Previously, we identified a vanadium-binding protein, Vanabin2, in which all 18 cysteines form nine disulfide bonds. Here, we report that Vanabin2 is a novel vanadium reductase because partial cleavage of its disulfide bonds results in the reduction of V(V) to V(IV). We propose that Vanabin2 forms a possible electron transfer cascade from the electron donor, NADPH, via glutathione reductase, glutathione, and Vanabin2 to the acceptor, and vanadium ions conjugated through thiol-disulfide exchange reactions.

Exclusive Expression of Transketolase in the Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea

Ascidians, especially those belonging to the Ascidiidae, are known to accumulate extremely high levels of vanadium in vanadocytes, one type of blood (coelomic) cell. Vanadium, which exists in the +5 oxidation state in seawater, is accumulated in the vanadocytes and reduced to the +3 oxidation state. We have been trying to characterize all of the polypeptides specific to vanadocytes and to specify the proteins that participate in the accumulation and reduction of vanadium. To date, we have localized three enzymes in vanadocytes: 6-phosphogluconate dehydrogenase (6-PGDH: EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49), and glycogen phosphorylase (GP: EC 2.4.1.1), all of which are involved in the pentose phosphate pathway. In the current study, we cloned a cDNA for transketolase, an essential and rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway, from vanadocytes. The cDNA encoded a protein of 624 amino acids, which showed 61.8% identity to the human adult-type transketolase gene product. By immunocytochemistry and immunoblot analyses, the transketolase was revealed to be a protein that was expressed only in vanadocytes and not in any of the more than ten other types of blood cell. This finding, taken together with the localized expression of the other three enzymes, strongly supports the hypothesis that the pentose phosphate pathway functions exclusively in vanadocytes.

Syntheses, structures, stability, and insulin-like activities of peroxovanadium(V) complexes with a heteroligand.

Several peroxovanadium(V) complexes were prepared with a tripodal or a quasi-tripodal tetradentate ligand. The structures of K(2)[VO(O(2))(nta)].2H(2)O and K[VO(O(2))(DL-cmhist)].H(2)O have been determined by X-ray crystallography (nta, nitrilotriacetate; cmhist, N-carboxymethylhistidinate). The structure of Cs[VO(O(2))(pda)].2H(2)O (pda, N-pyridylmethyliminodiacetate) has been estimated to be similar to that of K[VO(O(2))(DL-cmhist)].H(2)O. Each complex anion in these compounds adopts a distorted pentagonal bipyramidal structure, which is typical for heptacoordinate oxoperoxovanadium(V) complexes. The peroxide ion binds in a side-on fashion to the vanadium(V) center in the pentagonal plane. The peroxide anion in the cmhist complex dissociates rather easily in an acidic solution (pH approximately 3), while that in the other complexes stays intact under similar conditions. The in vitro insulin mimetic effect of the peroxovanadium(V) complexes has been evaluated by the inhibitory effect on free fatty acid (FFA) release in isolated rat adipocytes treated with epinephrine. The cmhist complex is effective, while the others are almost totally ineffective.

Glucose-6-Phosphate Dehydrogenase in the Pentose Phosphate Pathway Is Localized in Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea.

Abstract Ascidians are sessile marine animals known to accumulate high levels of vanadium selectively in vanadium-containing blood cells (vanadocytes). Almost all the vanadium accumulated in the vacuoles of vanadocytes is reduced to the +3 oxidation state via the +4 oxidation state, although vanadium is dissolved in the +5 oxidation state in sea water. Some of the reducing agents that participate in the reduction have been proposed. By chemical study, vanadium in the +5 oxidation state was reported to be reduced to the +4 oxidation state in the presence of NADPH. The present study revealed the existence of glucose-6-phosphodehydrogenase (G6PDH), the first enzyme to produce NADPH in the pentose phosphate pathway, in vanadocytes of a vanadium-rich ascidian. The results suggested that G6PDH conjugates the reduction of vanadium from the +5 through to the +4 oxidation state in vanadocytes of ascidians.

A 100-kDa Antigen Recognized by a Newly Prepared Monoclonal Antibody Specific to the Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea, is Glycogen Phosphorylase

Abstract Ascidians have the unusual physiological ability to accumulate high levels of vanadium and reduce it to the +3 oxidation state (VIII) in vanadocytes, the vanadium-containing blood cells. We are characterizing several polypeptides specific to vanadocytes that may participate in this. This study revealed that a 100-kDa antigen, recognized by a newly prepared monoclonal antibody, S8E4, is exclusively localized in vanadocytes, and identified the antigen as glycogen phosphorylase (EC 2.4.1.1) by sequencing the encoded cDNA. Since two enzymes, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), both in the pentose phosphate pathway, have already been identified in vanadocytes, at least three enzymes involved in carbohydrate metabolism are localized in vanadocytes in huge amounts.

ISOLATION OF CDNAS ENCODING SUBUNITS A AND B OF THE VACUOLAR-TYPE ATPASE FROM THE VANADIUM-RICH ASCIDIAN, ASCIDIA SYDNEIENSIS SAMEA

Abstract Vacuolar-type H+-ATPases (V-ATPases), which are composed of at least ten different subunits, can generate a proton-motive force by hydrolyzing ATP and acidify the contents of various intracellular organelles. Subunits A and B of V-ATPase have been detected immunologically in ascidian blood cells, predominantly in signet ring cells (vanadocytes), which accumulate vanadium in their vacuoles. The action of V-ATPase in ascidian blood cells has been demonstrated by the fact that bafilomycin A1, a specific inhibitor of V-ATPases, inhibits the acidification of the vacuoles of vanadocytes. As the next step in studying the function of V-ATPase in vanadocytes, we isolated cDNAs encoding subunits A and B of V-ATPase from the blood cells of an ascidian, Ascidia sydneiensis samea. The nucleotide sequences of the cDNAs for subunits A and B encoded proteins of 619 and 509 amino acids, respectively, both of which were highly conserved among organisms.

Extraction of a Vanadium-Binding Substance (Vanadobin) from the Blood Cells of Several Ascidian Species

A combination of techniques, including chromatography on Sephadex G-15 and SE-cellulose columns and neutron activation analysis for vanadium determination, was used to extract (at low pH) a vanadium-binding substance (vanadobin) from the blood cells of the ascidian species: Ascidia ahodori OKA, A. gemmata SLUITER, A. zara OKA, Corella japonica HERDMAN, and Ciona intestinalis (LINNE). In general, ascidians can be classified into two different categories based on vanadium content: species of the family Ascidiidae contain high levels of vanadium, whereas those in the Cionidae and Corellidae do not always have such high amounts. Because Ciona intestinalis and Corella japonica do have vanadobin in their blood cells, vanadobin may well be a universal complex in ascidians, having the role of accumulating vanadium in blood cells and maintaining its concentration. The blood cells of A. gemmata contained the highest amount of vanadium. Vanadobin extracted from these cells exhibits absorption spectra, not only in the ultraviolet region, but also in the visible region; such spectra correspond to those observed in vanadium complexes in oxidation states of +3 and +4.