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1 August 1998 Glucose-6-Phosphate Dehydrogenase in the Pentose Phosphate Pathway Is Localized in Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea
Taro Uyama, Kazuhiro Yamamoto, Kan Kanamori, Hitoshi Michibata
Author Affiliations +
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.

INTRODUCTION

High levels of vanadium, a transition metal, were first found by Henze (1911) in the blood cells (coelomic cells) of an ascidian known alternatively as a tunicate or seasquirt. Ever since, this unusual physiological phenomenon, never before reported in other organisms, has attracted the interest of investigators including not only physiologists but analytical, bioinorganic and biological chemists. To date, various studies have been done on this phenomenon as summarized in several review articles (Goodbody, 1974; Biggs and Swinehart, 1976; Kustin et al., 1983; Boyd and Kustin, 1985; Michibata, 1989, 1993, 1996; Michibata and Sakurai, 1990; Wever and Kustin, 1990; Smith et al., 1995; Kustin and Robinson, 1995; Michibata and Kanamori, 1998). The high levels of vanadium are exclusively contained in a type of blood cell, designated vanadocytes, one of approximately ten types of blood cells in ascidians (Michibata et al., 1987, 1991). The highest concentration of vanadium in vanadocytes exceeds 107 times the concentration in sea water (Michibata et al., 1991). Furthermore, almost all the vanadium accumulated in the vacuoles of vanadocytes is reduced to the +3 oxidation state (VIII), the most reduced form in aqueous solution (Lybing, 1953; Boeri and Ehrenberg, 1954; Webb, 1956; Carlson, 1975; Tullius et al., 1980; Dingley et al., 1981; Frank et al., 1986; Lee et al., 1988; Brand et al., 1989; Hirata and Michibata, 1991), although vanadium is reported to be dissolved in the +5 oxidation state (VV) in sea water (McLeod et al., 1975).

Some reducing agents must, therefore, participate in the accumulation process in vanadocytes. Several candidates for the reduction of vanadium in ascidian blood cells have been proposed. Tunichromes isolated from certain ascidian species (Bruening et al., 1985), glutathione, H2S, NADPH, dithiothreitol (Ryan et al., 1996), and thiol such as cysteine (Frank et al., 1987) have all been examined for their ability to reduce VV to VIV and/or VIV to VIII. However, not only has little direct evidence for involvement of these agents in the reduction in vanadocytes been obtained, but also no attention has been paid to whether these agents exist intrinsically in vanadocytes.

There is a good possibility that NADPH participates in the reduction of VV. Nour-Eldeen et al. (1985) reported that vanadate activates the catalysis via glucose-6-phosphate dehydrogenase of the oxidation of glucose by NADP+ in vitro. Shi and Dalal (1991, 1993) reported formation of VIV in the reduction of VV by NADPH-dependent flavoenzymes. It is known that 2 mols of NADPH are produced in the pentose phosphate pathway. One is produced by the reaction of glucose-6-phosphate dehydrogenase (G6PDH: EC1.1.1.49) and the other by that of 6-phosphogluconate dehydrogenase (6-PGDH: EC1.1.1.44).

The present experiment was therefore designed to examine whether G6PDH exists in ascidian blood cells, with the final aim being to prove the intrinsic participation of NADPH in the reduction of vanadium in the vanadocytes of ascidians. It was revealed immunocytologically that G6PDH was localized exclusively in vanadocytes and soluble extract of vanadocytes exhibited enzymatic activity of G6PDH.

MATERIALS AND METHODS

Ascidians

Specimens of the vanadium-rich ascidian, Ascidia sydneiensis samea, were collected in the vicinity of the Asamushi Marine Biological Station of Tohoku University at Asamushi, Aomori Prefecture, and of the Otsuchi Marine Research Center, Ocean Research Institute, the University of Tokyo, Otsuchi, Iwate Prefecture, Japan. The ascidians were maintained in an aquarium that contained circulating natural sea water at 18°C.

Immunocytological detection

To examine the localization of G6PDH, immunological detection was carried out in a similar manner to that described previously (Uyama et al., 1991, 1994). Ascidian blood, drawn by making an incision through the lower part of the tunic and puncturing the heart at 4°C, was suspended in Ca2+- and Mg2+-free artificial sea water containing, 460 mM NaCl, 9 mM KCl, 32 mM Na2SO4, 6 mM NaHCO3 and 5 mM HEPES at pH 7.0 to avoid clotting of the blood cells and centrifuged at 300 × g for 10 min at 4°C to separate the blood cells from the serum. The blood cells were resuspended in Ca2+- and Mg2+-free artificial sea water and were mounted on coverslips. The coverslips were immersed in ethanol containing 5% formalin for 5 min at −15°C. After fixation, the coverslips were washed with phosphate buffered saline (PBS), which consisted of 136.9 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, at pH 7.2, for 30 min at room temperature. Next, they were immersed first in 100 μl of 10% goat normal serum for 1 hr at room temperature to reduce the non-specific background, then in 100 μl of anti-G6PDH antibody raised in rabbit against G6PDH of bakers yeast (Sigma Chemical Co., St. Louis, USA) which had been diluted at a ratio of 1 to 1000 with PBS containing 10% goat normal serum for 1 hr at room temperature. The coverslips were then washed with PBS for 30 min, before being immersed in 100 μl of fluorescein isothiocyanate-conjugated antiserum raised in goat against rabbit IgG (Organon Teknika Corporation, Philadelphia, USA) which was diluted at a ratio of 1 to 2000 with PBS for 1 hr at room temperature. Finally, they were washed with PBS for 1 hr, mounted in 80% glycerol, and observed under a microscope (Olympus Co., Ltd., Tokyo) equipped with an epifluorescence optics unit. Both bright field and fluorescence photographs of the blood cells were taken with Fuji color film (ASA 400). As a negative control, a few cover slips were immersed in preimmune rabbit serum in the same manner. Vanadocytes were identified not only by morphological appearance but by the immunoreactivity with S4D5 monoclonal antibody, specific to vanadocytes (Michibata et al., 1987, 1990; Uyama et al., 1991).

Western blot analysis

G6PDH is known to be a soluble protein composed of a dimer of identical subunit with Mrof 50-60 kDa (Takizawa et al., 1986; Camardella et al., 1988; Jeffery et al., 1989; Persson et al., 1991). Therefore, to examine whether the enzyme exists in a soluble protein fraction extracted from ascidian blood cells, Western blot analysis was applied using the antiserum against G6PDH. An aliquot of 200 mg wet weight of blood cells of A. sydneiensis samea was homogenized in 6 ml of 0.2 M Tris-HCl buffer at pH 8.0 containing protease inhibitors [leupeptin, pepstatin A, chymostatin, phenylmethylsulfonyl fluoride (PMSF), each at a concentration of 10 μg/ml] using a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 100,000 × g for 1 hr. The supernatant was collected and used as soluble protein for Western blot analysis and enzymatic assay of G6PDH. An aliquot of approx. 30 μg of the soluble protein was dissolved in a sample dissociation buffered solution consisting of 62.5 mM Tris-HCl at pH 6.8, 5%(v/v) 2-mercaptoethanol, 10%(v/v) glycerol and 2.3% (w/v) SDS. As the positive control, a purified G6PDH of the bakers yeast (Sigma) was purchased and an aliquot of 100 ng of the protein was used in the same manner. The protein content in each sample was determined by the Bradford (1976) method using a Bio-Rad Protein Assay kit (Nippon Bio-Rad Laboratories, Inc., Tokyo, Japan) and bovine serum albumin as a standard. Each sample was subjected to electrophoresis in a 10% polyacrylamide gel in the presence of 2% SDS. The proteins separated on SDS-PAGE electrophoretically were blotted onto a nitrocellulose paper for Western blot analysis as described previously (Uyama et al., 1997; Kanda et al., 1997).

Enzymatic assay

G6PDH activity in the 100,000 × g supernatant obtained from ascidian blood cells was assayed, since it was confirmed that an antigen recognized by the anti-G6PDH existed in the supernatant. Two kinds of reaction mixtures were prepared. One consisted of glucose 6-phosphate and 6-phosphogluconate ranging in concentrations from 10 μM to 200 μM (Sigma) as substrates, 0.4 mM NADP+ (Oriental Yeast Co., LTD), and 5 mM MgCl2 in 0.2 M Tris-HCl buffer solution at pH 8.0. The other did not contain glucose 6-phosphate. The reaction was initiated by the addition of 50 μl of enzyme solution (containing approx. 50 μg protein). Thus the final volume of the reaction mixture was 2.5 ml. Reduction of NADP+ to NADPH as a result of the enzymatic reaction was recorded as the increase of absorbance at 340 nm. G6PDH activity was calculated by subtracting 6-PGDH activity from G6PDH plus 6-PGDH activities. Protein concentration was determined as described above to calculate the specific activity.

RESULTS

Immunocytological detection of G6PDH

As shown in Fig. 1, immunoreactivity of anti-G6PDH antibody was detected only in signet ring cells which had been identified as vanadocytes (Michibata et al., 1987, 1990). Although A. sydneiensis samea has at least six different types of blood cells (Michibata et al., 1990; Kaneko et al., 1995; Wuchiyama and Michibata, 1995), no immunoreactivity was observed in blood cells other than vanadocytes.

Fig. 1

Immunocytological detection of G6PDH in the vanadocytes of the vanadium-rich ascidian, Ascidia sydneiensis samea. Blood cells observed in panels A and a were reacted with anti-G6PDH antibody. Blood cells in panels B and b were reacted with preimmune rabbit serum as a negative control. Upper panels (A and B) and lower panels (a and b) were visualized by a Nomarski differential-interference and by fluorescence microscopy, respectively. Vanadocytes (signet ring cells) were exclusively recognized with anti-G6PDH antibody, showing fluorescence of FITC. No immunoreactivity was observed in the other types of blood cells. s, vanadocytes (signet ring cells). Scale bar indicates 10 μm.

i0289-0003-15-4-441-f01.jpg

Western blot analysis of G6PDH

Western blot analysis with anti-G6PDH antibody revealed a positive band of 58 kDa in soluble proteins, as shown in Fig. 2. The corresponding molecular mass of 58 kDa is in good agreement with that previously reported in other living organisms (Takizawa et al., 1986; Camardella et al., 1988; Jeffery et al., 1989; Persson et al., 1991). This result demonstrates that G6PDH exists in the soluble protein fraction of the ascidian blood cells.

Fig. 2

Localization of a 58-kDa antigen in ascidian soluble fraction revealed by Western blot analysis using anti-G6PDH antibody. Blood cells of Ascidia sydneiensis samea were homogenized and proteins in the soluble fraction were separated by SDS-PAGE and visualized by staining with Coomassie brilliant blue (left). The separated proteins were blotted onto a nitrocellulose paper and reacted with anti-G6PDH antibody to examine whether G6PDH is present in the ascidian blood cells (right). A positive band of 58 kDa was detected in the soluble fraction, as well as in yeast G6PDH. This revealed that G6PDH exists in the soluble protein fraction of the ascidian blood cells. Lane 1, the soluble protein (30 μg) extracted ascidian blood cells; Lane 2, a purified G6PDH of the bakers yeast (100 ng protein).

i0289-0003-15-4-441-f02.gif

Enzymatic assay of G6PDH

To examine whether the enzymatic activity of G6PDH is actually present in soluble extract of the blood cells, the soluble extract was assayed using glucose-6-phosphate as a substrate. A correspondingly high level of enzymatic activity of G6PDH was detected. The Lineweaver-Burk plot shows clearly that the enzymatic activity is dependent on the concentration of substrate (Fig. 3). Km for the substrate and Vmax were 99.2 μmol/l and 196 nmol/min, respectively, at pH 7.4.

Fig. 3

Enzymatic activity of G6PDH in the ascidian blood cells. To examine whether G6PDH functions enzymatically in the soluble protein extracted from ascidian blood cells containing vanadocytes, the activity was assayed using glucose-6-phosphate (G6P). The Lineweaver-Burk plot shows clearly that the enzymatic activity is dependent on the concentration of substrate. Km for the substrate and Vmax are 99.2 μmol/l and 196 nmol/min, respectively.

i0289-0003-15-4-441-f03.gif

DISCUSSION

The present experiments have revealed that G6PDH, the first enzyme in the pentose phosphate pathway producing 6-phosphoglucono-δ-lactone and reducing NADP+ to NADPH, is present in vanadocytes, vanadium-containing blood cells, of the vanadium-rich ascidian Ascidia sydneiensis samea. Since immunoreactivity of anti-G6PDH antibody was observed in vanadocytes but not in other types of blood cells (Fig. 1), it is clear that G6PDH is localized in vanadocytes. G6PDH was further found to be localized in the cytoplasm and not in the vacuoles of vanadocytes on close observation. By Western blot analysis anti-G6PDH antibody was revealed to have reactivity with a 58 kDa protein in the soluble fraction obtained by centrifugation at 100,000 × g (Fig. 2). Furthermore, a correspondingly high level of enzymatic activity of G6PDH was found in the soluble fraction of the blood cells (Fig. 3).

Vanadocytes, having high levels of vanadium, sulfate ions and protons in their vacuoles (Michibata et al., 1991; Kanamori and Michibata, 1994; Uyama et al., 1994), are not found in other living organisms. Under these conditions, vanadium is kept in the VIII form, the most reduced form in aqueous solution (Hirata and Michibata, 1991). Some reducing and/or chelating agents must participate in the reduction and protection against air-oxidation in vanadocytes. In fact, several candidates have been proposed, such as haemovanadin (Califano and Boeri, 1950; Webb, 1956) and tunichromes (Bruening et al., 1985). However, the involvement of these compounds in reduction of vanadium is unclear (Kime-Hunt et al., 1988; Michibata et al., 1988, 1990; Bulls et al., 1990; Tsuchida et al., 1994; Ryan et al., 1996).

Recently, we have revealed that 6-phosphogluconate dehydrogenase (6-PGDH), the third enzyme of the pentose phosphate pathway, was localized in vanadocytes of the ascidian using immunological methods, that the full-length cDNA encoding 6-GDH was isolated and that soluble extract of the blood cells further exhibited a correspondingly high level of 6-PGDH enzymatic activity (Uyama et al., 1998). The pentose phosphate pathway is the major supplier of reducing agents in the form of NADPH and is tightly coupled to cellular processes which require NADPH and other reductase systems. It has been reported that VV stimulates oxidation of NAD(P)H; namely, VV is reduced to VIV in the presence of NAD(P)H in vitro. Erdmann et al. (1979) first noted that VV stimulated the oxidation of NADH by plasma membranes and attributed this effect to a membrane-containing NAD(P)H-dependent VV reductase. Liochev and Fridovich (1990) proposed that NAD(P)H dehydrogenases or oxidases produce O2, which causes VV to stimulate NAD(P)H oxidation and endogenous superoxide plays a central role in this reaction. Shi and Dalal (1991, 1993) demonstrated that O2 radicals are not significantly involved in the VIV generation but they pointed out that VIV is generated in the microsomal reduction of VV in the presence of NAD(P)H and the VIV formation exhibits typical enzymatic kinetics. In fact, our preliminary data showed that NADPH can reduce VV to VIV in vitro (to be published elsewhere). These observations suggest that NADPH conjugates the reduction of VV to VIV in the vanadocytes of ascidians, although there is controversy as to the mechanism involved. While almost all vanadium ions stored in the vacuoles of vanadocytes are further reduced to VIII, no reducing agents that can reduce VV or VIV to VIIIhave been extracted from ascidian blood cells to date.

The abbreviations used are

HEPES

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

EDTA

ethylenediaminetetraacetic acid

Tris

tris (hydroxymethyl) aminomethane

NADPH

nicotinamide adenine dinucleotide phosphate reduced form

NADP+

nicotinamide adenine dinucleotide phosphate oxidized form.

Acknowledgments

The authors express their heartfelt thanks to the staff of Asamushi Marine Biological Station of Tohoku University, Aomori Prefecture and of the Otsuchi Marine Research Center, Ocean Research Institute of the University of Tokyo, Iwate Prefecture, Japan. Thanks are also due to Mr. N. Abo in our laboratory who collected some of the animals and kept them healthy in an aquarium. This work was partially supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (#09440278, #09874178 and #09839017).

REFERENCES

1.

W. R. Biggs and J. H. Swinehart . 1976. Vanadium in selected biological systems. In “Metal Ions in Biological Systems Vol 6”. Ed by H. Sigel , editor. Marcel Dekker. New York. pp. 141–196. Google Scholar

2.

E. Boeri and A. Ehrenberg . 1954. On the nature of vanadium in vanadocytes hemolysate from ascidians. Arch Biochem Biophys 50:404–416. Google Scholar

3.

D. W. Boyd and K. Kustin . 1985. Vanadium: A versatile biochemical effector with an elusive biological function. Adv Inorg Biochem 6:311–365. Google Scholar

4.

M. M. Bradford 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein in utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. Google Scholar

5.

S. G. Brand, C. J. Hawkins, A. T. Marshall, G. W. Nette, and D. L. Parry . 1989. Vanadium chemistry of ascidians. Comp Biochem Physiol 93B:425–436. Google Scholar

6.

R. C. Bruening, E. M. Oltz, J. Furukawa, K. Nakanishi, and K. Kustin . 1985. Isolation and structure of tunichrome B-1, a reducing blood pigment from the tunicate Ascidia nigra L. J Am Chem Soc 107:5298–5300. Google Scholar

7.

A. R. Bulls, C. G. Pippin, F. E. Hahn, and K. N. Raymond . 1990. Synthesis and G6PDH in Vanadocytes of Ascidian 445 characterization of a series of vanadium-tunichrome B1 analogues. Crystal structure of a tris(catecholamide) complex of vanadium. J Am Chem Soc 112:2627–2632. Google Scholar

8.

L. Califano and E. Boeri . 1950. Studies on haemovanadin. III. Some physiological properties of haemovanadin, the vanadium compound of the blood of Phallusia mammillata Cuv. J Exp Zool 27:253–256. Google Scholar

9.

L. Camardella, C. Caruso, B. Rutigliano, M. Romano, G. Di Prisco, and F. Descalzi-Cancedda . 1988. Human erythrocyte glucose-6-phosphate dehydrogenase. Identification of a reactive lysyl residue labelled with pyridoxal 5′-phoaphate. Eur J Biochem 171:485–489. Google Scholar

10.

R. M. K. Carlson 1975. Nuclear magnetic resonance spectrum of living tunicate blood cells and the structure of the native vanadium chromogen. Proc Natl Acad Sci USA 72:2217–2221. Google Scholar

11.

A. L. Dingley, K. Kustin, I. G. Macara, and G. C. McLeod . 1981. Accumulation of vanadium by tunicate blood cells occurs via a specific anion transport system. Biochim Biophys Acta 649:493–502. Google Scholar

12.

E. Erdmann, W. Kraweitz, G. Philipp, I. Hackbarth, W. Schmitz, H. Scholtz, and F. L. Crane . 1979. Purified cardiac cell membranes with high (Na+ + K+) ATPase activity contain significant NADH-vanadate reductase activity. Nature 282:335–336. Google Scholar

13.

P. Frank, R. M. K. Carlson, and K. O. Hodgson . 1986. Vanadyl ion EPR as a non-invasive probe of pH in intact vanadocytes from Ascidia ceratodes. Inorg Chem 25:470–478. Google Scholar

14.

P. Frank, B. Hedman, R. K. Carlson, T. A. Tyson, A. L. Row, and K. O. Hodgson . 1987. A large reservoir of sulfate and sulfonate resides within plasma cells from Ascidia ceratodes, revealed by X-ray absorption near-edge structure spectroscopy. Biochemistry 26:4975–4979. Google Scholar

15.

I. Goodbody 1974. The physiology of ascidians. Adv Mar Biol 12:1–149. Google Scholar

16.

M. Henze 1911. Untersuchungen über das Blut der Ascidien. I. Mitteilung. Die Vanadiumverbindung der Blutkörperchen. HoppeSeyler's Z Physiol Chem 72:494–501. Google Scholar

17.

J. Hirata and H. Michibata . 1991. Valency of vanadium in the vanadocytes of Ascidia gemmata separated by density-gradient centrifugation. J Exp Zool 257:160–165. Google Scholar

18.

J. Jeffery, J. Barros-Söderling, L. Murray, I. Wood, R. Hansen, B. Szepesi, and H. Jörnvall . 1989. Glucose-6-phosphate dehydrogenase. Characteristics revealed by the rat liver enzyme structure. Eur J Biochem 186:551–556. Google Scholar

19.

K. Kanamori and H. Michibata . 1994. Raman spectroscopic study of the vanadium and sulphate in blood cell homogenates of the ascidian, Ascidia gemmata. J Mar Biol Ass UK 74:279–286. Google Scholar

20.

K. Kanda, Y. Nose, J. Wuchiyama, T. Uyama, Y. Moriyama, and H. Michibata . 1997. Identification of a vanadium-associated protein from the vanadium-rich ascidian, Ascidia sydneiensis samea. Zool Sci 14:37–42. Google Scholar

21.

A. Kaneko, T. Uyama, Y. Moriyama, and H. Michibata . 1995. Localization, with monoclonal antibodies and by detection of autonomous fluorescence, of blood cells in the tissues of the vanadium-rich ascidian, Ascidia sydneiensis samea. Zool Sci 12:733–739. Google Scholar

22.

E. Kime-Hunt, K. Spartalian, and C. J. Carrano . 1988. Models for vanadiumtunichrome interactions. J Chem Soc Chem Commun 1217–1218. Google Scholar

23.

K. Kustin, G. C. McLeod, T. R. Gilbert, and L. B. R. Briggs 4th . 1983. Vanadium and other metal ions in the physiological ecology of marine organisms. Structure and Bonding 53:139–160. Google Scholar

24.

K. Kustin and W. E. Robinson . 1995. Vanadium transport in animal systems. In “Metal ions in Biological Systems Vol 31”. Ed by H. Sigel and A. Sigel , editors. Marcel Dekker. New York. pp. 511–542. Google Scholar

25.

S. Lee, K. Kustin, W. E. Robinson, R. B. Frankel, and K. Spartalian . 1988. Magnetic properties of tunicate blood cells. I. Ascidia nigra. Inorg Biochem 33:183–192. Google Scholar

26.

S. I. Liochev and I. Fridovich . 1990. Vanadate-stimulated oxidation of NAD(P)H in the presence of biological membranes and other sources of O2. Arch Biochem Biophys 279:1–7. Google Scholar

27.

S. Lybing 1953. The valence of vanadium in hemolysates of blood from Ascidia obliqua Alder. Arkiv Kemi 6:261–269. Google Scholar

28.

G. C. McLeod, K. V. Ladd, K. Kustin, and D. L. Toppen . 1975. Extraction of vanadium(V) from seawater by tunicate: A revision of concepts. Limnol Oceanograph 20:491–493. Google Scholar

29.

H. Michibata 1989. New aspects of accumulation and reduction of vanadium ions in ascidians, based on concerted investigation for both a chemical and biological viewpoint. Zool Sci 6:639–681. Google Scholar

30.

H. Michibata 1993. The mechanism of accumulation of high levels of vanadium by ascidians from seawater: Biophysical approaches to a remarkable phenomenon. Adv Biophys 29:103–131. Google Scholar

31.

H. Michibata 1996. The mechanism of accumulation of vanadium by ascidians: Some progress towards an understanding of this unusual phenomenon. Zool Sci 13:489–502. Google Scholar

32.

H. Michibata, J. Hirata, M. Uesaka, T. Numakunai, and H. Sakurai . 1987. Separation of vanadocytes: Determination and characterization of vanadium ion in the separated blood cells of the ascidian, Ascidia ahodori. J Exp Zool 244:33–38. Google Scholar

33.

H. Michibata, J. Hirata, T. Terada, and H. Sakurai . 1988. Autonomous fluorescence of ascidian blood cells with special reference to identification of vanadocytes. Experientia 44:906–907. Google Scholar

34.

H. Michibata and H. Sakurai . 1990. Vanadium in ascidians. In “Vanadium in Biological Systems”. Ed by N. D. Chasteen , editor. Kluwer Acad Publ. Dortrecht. pp. 153–171. Google Scholar

35.

H. Michibata, T. Uyama, and J. Hirata . 1990. Vanadium containing cells (vanadocytes) show no fluorescence due to the tunichrome in the ascidian Ascidia sydneiensis samea. Zool Sci 7:55–61. Google Scholar

36.

H. Michibata, Y. Iwata, and J. Hirata . 1991. Isolation of highly acidic and vanadium-containing blood cells from among several types of blood cell from Ascidiidae species by density gradient centrifugation. J Exp Zool 257:306–313. Google Scholar

37.

H. Michibata and K. Kanamori . 1998. Selective accumulation of vanadium by ascidians from sea water. In “Vanadium in the Environment. Part one: Chemistry and Biochemistry”. Ed by J. O. Nriagu , editor. John Wiley & Sons, Inc. New York. pp. 217–249. Google Scholar

38.

A. F. Nour-Eldeen, M. M. Craig, and M. J. Gresser . 1985. Interaction of inorganic vanadate with glucose-6-phosphate dehydrogenase. J Biol Chem 260:6836–6842. Google Scholar

39.

B. Persson, H. Jörnvall, I. Wood, and J. Jeffery . 1991. Functionally important regions of glucose-6-phosphate dehydrogenase defined by the Saccharomyces cerevisiae enzyme and its differences from the mammalian and insect forms. Eur J Biochem 198:485–491. Google Scholar

40.

D. E. Ryan, K. B. Grant, K. Nakanishi, P. Frank, and K. O. Hodgson . 1996. Reactions between vanadium ions and biogenic reductants of tunicates: Spectroscopic probing for complexation and redox products in vitro. Biochemistry 35:8651–8661. Google Scholar

41.

X. Shi and N. S. Dalal . 1991. Flavoenzymes reduce vanadium(V) and molecular oxygen and generate hydroxyl radical. Arch Biochem Biophys 289:355–361. Google Scholar

42.

X. Shi and N. S. Dalal . 1993. One-electron reduction of vanadium(V) by flavoenzymes/NADPH. Arch Biochem Biophys 302:300–303. Google Scholar

43.

M. J. Smith, D. E. Ryan, K. Nakanishi, P. Frank, and K. O. Hodgson . 1995. Vanadium in ascidians and the chemistry of tunichromes. In “Metal ions in Biological Systems Vol 31”. Ed by H. Sigel and A. Sigel , editors. Marcel Dekker. New York. pp. 423–490. Google Scholar

44.

T. Takizawa, I-Y. Huang, T. Ikuta, and A. Yoshida . 1986. Human glucose-6-phosphate dehydrogenase: Primary structure and cDNA cloning. Proc Natl Acad Sci USA 83:4157–4161. Google Scholar

45.

E. Tsuchida, K. Yamamoto, K. Oyaizu, N. Iwasaki, and F. C. Anson . 1994. Electrochemical investigations of the complexes resulting from the acid-promoted deoxygenation and dimerization of (N,N′-ethylenebis (salicylideneaminato) oxovanadium(IV). Inorg Chem 33:1056–1063. Google Scholar

46.

T. D. Tullius, W. O. Gillum, R. M. K. Carlson, and K. O. Hodgson . 1980. Structural study of the vanadium complex in living ascidian blood cells by X-ray absorption spectrometry. J Am Chem Soc 102:5670–5676. Google Scholar

47.

T. Uyama, T. Nishikata, N. Satoh, and H. Michibata . 1991. Monoclonal antibody specific to signet ring cells, the vanadocytes of the tunicate, Ascidia sydneiensis samea. J Exp Zool 259:196–201. Google Scholar

48.

T. Uyama, Y. Moriyama, M. Futai, and H. Michibata . 1994. Immunological detection of a vacuolar-type H+-ATPase in the vanadocytes of the ascidian Ascidia sydneiensis samea. J Exp Zool 270:148–154. Google Scholar

49.

T. Uyama, Y. Nose, J. Wuchiyama, Y. Moriyama, and H. Michibata . 1997. Finding of the same antigens in the polychaeta, Pseudopotamilla occelata, as those in the vanadium-rich ascidian, Ascidia sydneiensis samea. Zool Sci 14:43–47. Google Scholar

50.

T. Uyama, T. Kinoshita, H. Takahashi, N. Satoh, K. Kanamori, and H. Michibata . 1998. 6-Phosphogluconate dehydrogenase is a 45-kDa antigen recognized by S4D5, a monoclonal antibody specific to vanadocytes in the vanadium-rich ascidian Ascidia sydneiensis samea. J Biochem (in press). Google Scholar

51.

D. A. Webb 1956. The blood of tunicates and the biochemistry of vanadium. Publ Staz Zool Napoli 28:273–288. Google Scholar

52.

R. Wever and K. Kustin . 1990. Vanadium: A biologically relevant element. In “Advances in Inorganic Chemistry Vol 35”. Ed by A. G. Sykes , editor. Academic Press. New York. pp. 81–115. Google Scholar

53.

J. Wuchiyama and H. Michibata . 1995. Classification, based on autonomous fluorescence, of the blood cells of several ascidians that contain high levels of vanadium. Acta Zool (Stockholm) 76:51–55. Google Scholar
Taro Uyama, Kazuhiro Yamamoto, Kan Kanamori, and Hitoshi Michibata "Glucose-6-Phosphate Dehydrogenase in the Pentose Phosphate Pathway Is Localized in Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea," Zoological Science 15(4), 441-446, (1 August 1998). https://doi.org/10.2108/0289-0003(1998)15[441:GDITPP]2.0.CO;2
Received: 5 March 1998; Accepted: 1 April 1998; Published: 1 August 1998
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