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.
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.
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.
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.
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).