The Accumulation Mechanism of Vanadium by Ascidians - ACS

Chapter 19

The Accumulation Mechanism of Vanadium by Ascidians

Downloaded by CORNELL UNIV on May 11, 2017 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch019

An Interdisciplinary Study between Biology and Chemistry on Extraordinary High Levels and Reduced Form of Vanadium in Vanadocytes 1

1

2

H. Michibata , T. Uyama , and K. Kanamori 1

Marine Biological Laboratory, Faculty of Science and Laboratory of Marine Molecular Biology, Graduate School of Science, Hiroshima University, Mukaishima-cho, Hiroshima 722, Japan Department of Chemistry, Faculty of Science, Toyama University, Gofuku, Toyama 930, Japan

2

The unusual phenomenon whereby some ascidians, known as tunicates, accumulate high levels of vanadium in their blood cells has attracted the interest of many investigators. The highest concentration of vanadium, 350 mM, in vanadocytes corresponds 10 times higher than that in sea water and vanadium is stored in the +3 oxidation state. Enzymes in the pentose phosphate pathway in vanadocytes have a possibility to participate the reduction of vanadium. The content of the vacuole of signet ring cells was kept to be highly acidic, pH 1.9 to 4.2, by the function of a vacuolar type H -ATPase. A vanadium-associated protein (VAP) was isolatedfromthe blood cells and a monoclonal antibody against VAP was raised. Biochemical characterization of VAP and cloning of the gene encoded VAP are in progress. Ascidians, known as tunicates or sea squirts, are phylogenically classified into the phylum Chordata between Invertebrata and Vertebrata. A German chemist, Martin Henze discovered high levels of vanadium in the blood cells (coelomic cells) of an ascidian collected from the Bay of Naples (Henze, 1911). His finding attracted not only analytical chemists, but also physiologists, biochemists, and chemists of natural products, in part because of the initial interest in the extraordinary high level of vanadium never before reported in other organisms but also because of the considerable interest in the possible participation of vanadium in oxygen transport as a third candidate in addition to iron and copper. After the first finding of vanadium in ascidian blood cells, many species of ascidians were analyzed for presence of vanadium. The data obtained vary widely in sensitivity and in precision as do data on dry weight, wet weight, ash weight, or amount of protein. Re-determination of the contents of vanadium was, therefore, required. 7

+

248

©1998 American Chemical Society Tracey and Crans; Vanadium Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

249 Many species of ascidians belonging to two of three suborders, Phlebobranchia and Stolidobranchia were collected by ourselves mainly from the waters around Japan and the Mediterranean. Samples of blood cells, plasma, tunic, mantle (muscle), branchial basket, stomach, hepatopancreas, and gonad were submitted to neutron-activation analysis in a nuclear reactor, which was an extremely sensitive method for the quantification of vanadium (Michibata, 1984; Michibata et al., 1986). The data obtained are summarized in Table 1. Although vanadium was detectable in samples from almost all species examined, the ascidian species belonging to the suborder Phlebobranchia apparently contained higher levels of vanadium than those belonging to Stolidobranchia. We also confirmed that blood cells contained the highest amounts of vanadium among the tissues examined. Furthermore, the highest concentration of vanadium, 350 mM (mmol/dm ) was found in the blood cells of Ascidia gemmata belonging to the suborder Phlebobranchia (Michibata etal., 1991a); 350 m M corresponds 10 times the vanadium concentration in sea water (Cole etal., 1983; Collier, 1984). Blood cells were confirmed to contain the highest amounts of vanadium among the tissues examined in ascidians. Ascidian blood cells are classified into nine to eleven different types that can be grouped into six categories on the basis of their morphology: hemoblasts, lymphocytes, leucocytes, vacuolated cells, pigment cells and nephrocytes (Wright, 1981). The vacuolated cells can be further divided into at least four different types: morula cells, signet ring cells, compartment cells and small compartment cells. For many years, the morula cells were thought to be the so-called vanadocytes (Webb, 1939; Endean, 1960; Kalk, 1963a, b; Kustin etal., 1976), because their pale-green color resembles that of a vanadium complex dissolved in aqueous solution and the dense granules in morula cells, which can be observed under an electron microscope after fixation with osmium tetroxide, were assumed to be deposits of vanadium. At the end of the 1970's, with the increasing availability of scanning transmission electron microscopes equipped with an energy disperse X-ray detector, it became possible to address with greater confidence the question of whether morula cells are the true vanadocytes. A n Italian group was the first to demonstrate that the characteristic X-ray due to vanadium was not detected from morula cells but from granular amoebocytes, signet ring cells and compartment cells and, moreover, that vanadium was selectively concentrated in the vacuolar membranes of these cells where vanadium granules was present inside the vacuoles (Botte etal., 1979a; Scippa etal., 1982, 1985; Rowley, 1982). Identification of the true vanadocytes became a matter of the highest priority to those concerned with the mechanism of accumulation of vanadium by ascidians. To end the controversy and identify the true vanadocytes, we used a combination of density gradient centrifugation, for the isolation of specific types of blood cells and neutron-activation analysis, for the quantification of the vanadium contents of the isolated subpopulations of blood cells (Michibata etal., 1987). The subpopulation of blood cells recovered from each layer was submitted to


3

7

Tracey and Crans; Vanadium Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


250 neutron-activation analysis. The distribution pattern of vanadium coincided with that of signet ring cells but not with that of morula cells or compartment cells. These results proved that the signet ring cells are the true vanadocytes (Fig. 1) (Michibata et al., 1987, 1991a; Hirata and Michibata, 1991). The establishment of reliable cell markers for the recognition of different types of blood cells is necessary to clarify not only the function but also the lineage of each type of cell. We prepared a monoclonal antibody, which we hoped might serve as a powerful tool for solving these problems, using a homogenate of the subpopulation of signet ring cellsfromAscidia sydneiensis samea as the antigen (Uyama etal., 1991). The monoclonal antibody obtained, S4D5, was shown to react specifically with the vanadocytes not only from A. sydneiensis samea but also from two additional species, A. gemmata and A. ahodori. Immunoblotting analysis showed that this antibody recognizes a single polypeptide of about 45 kDa from all three species. Henze (1911) was the first to suggest the existence of vanadium in the +5 oxidation state. Later, many workers reported the +3 oxidation state of vanadium. More recently, studies using noninvasive physical methods, including ESR, X A S , N M R , and SQUID, indicated that the vanadium ions in ascidian blood cells were predominantly in the +3 oxidation state, with a small amount being in the +4 oxidation state (Carlson, 1975; Tullius etal., 1980; Dingley etal., 1981; Frank etal., 1986; Lee etal., 1988; Brand etal., 1989). These results were, however, derived not from the vanadocytes but from the entire population of blood cells. Thus, some questions remained to be answered. In particular, does vanadium exist in two oxidation states in one type of blood cell, or is each state formed in a different cell type? After separation of the various types of blood cells of A. gemmata, we made noninvasive ESR measurements of the oxidation state of vanadium in the fractionated blood cells under a reducing atmosphere (Hirata and Michibata, 1991). Consequently, it was revealed that vanadium in vanadocytes is predominantly in the +3 oxidation state, with a small amount being in the +4 oxidation state. The ratio of vanadium(m) to vanadium(IV) was 97.6:2.4 (Table 2). A considerable amount of sulfate has always been found in association with vanadium in ascidian blood cells (Henze, 1932; Califano etal, 1950; Bielig et al., 1954; Levine, 1961; Botte etal., 1979a, b; Scippa etal., 1982; 1985; 1988; Bell etal, 1982; Pirie era/., 1984; Lane etal, 1988; Frank etal, 1986, 1987, 1994, 1995; Anderson and Swinehart, 1991), suggesting that sulfate might be involved in the biological function and/or the accumulation and reduction of vanadium. Raman spectroscopy can be also used to detect sulfate ion selectively in ascidian blood cells because sulfate ion gives a very intense Raman band at the diagnostic position, 983 c m . We observed fairly good Raman spectrum of the blood cell lysate from Ascidia gemmata, which has the highest concentration of vanadium(IU) among ascidians (Kanamori and Michibata, 1994). Vanadium(DI) ions in the blood cells were converted to vanadyl(IV) ions by air-oxidation prior to Raman measurements so as to facilitate detection based on V=0 stretching vibration. From analysis of -1



251

Figure 1. Morula cells (A), misidentified initially as vanadocytes, and signet ring cells (B), identified newly as vanadocytes, in the ascidian, Ascidia ahodori. Scale bar indicates 10 μηι. (Michibata etal.,J. Exp. Zool., 1987, 244: 33-38.)


252


Table 1. Concentrations of Vanadium in Tissues of Several Ascidians (mM) Tunic Mantle Branchial Serum Blood cells basket Phlebobranchia 347.2 N.D. Ascidia gemmata N.D. N.D. N.D. 59.9 1.0 A. ahodori 2.4 11.2 12.9 12.8 0.05 A. sydneiensis 0.06 0.7 1.4 19.3 Phallusia mammillata N.D. 0.03 2.9 0.9 0.6 0.008 Ciona intestinalis 0.003 0.7 0.7 Stolidobranchia Styela plicata 0.005 Halocynthia roretzi0.01 H. aurantium 0.002

0.001 0.001 0.002

0.001 0.004 0.002

0.003 0.001 N.D.

0.007 0.007 0.004

N.D.: not determined. (Michibata etal, Biol. Bull., 1986, 171: 672-681.; Michibata etal., J. Exp. Zool, 1991, 257: 306-313.) Table 2. Vanadium Spceies and Ratio between Vanadium and Sulfate in Ascidian Blood Cells V : V 0 = 98 : 2 V : S 0 ~ = 1:1.5 3 +

2 +

3 +

2

4

(Michibata etal., J. Exp. Zool, 1991, 257: 306-313.; Kanamori, K. and H. Michibata, J. Mar. Biol. Ass. U.K., 1994, 74: 279-286.)

2+

2

the band intensities due to V = 0 and S 0 ~ ions, we estimated the content ratio of sulfate to vanadium to be approximately 1.5, as would be predicted if sulfate ions were present as the counter ions of vanadium(IQ) (Table 2). Carlson (1975) reported a similar value of the content ratio for Ascidia ceratodes, but lower values were obtained by Bell etal. (1982) and Frank etal. (1986). The monoclonal antibody, designated S4D5, specific to vanadocytes was revealed to react with a single polypeptide of about 45 kDa extracted from not only A. sydneiensis samea which offered the antigen but also the other vanadium-rich ascidians, A. ahodori, A. gemmata and Ciona intestinalis. The antigen of 45 kDa peptide was further disclosed to be localized in the cytoplasmic region and around the intravacuolar vesicle of the vanadocytes by immunocytological studies. This polypeptide is predominant among many peptides extracted from a subpopulation of vanadocytes in A. sydneiensis samea. However, the function of the polypeptide is not known. The recent experiments demonstrate that the antigen of 45 kDa localized in vanadocytes is 6-phosphogluconate dehydrogenase (6PGDH: EC1.1.1.44) which is an enzyme of the pentose phosphate pathway, based on cDNA isolation of R N A samples from blood cells of the ascidian. Soluble 4



253 extract of vanadocytes further exhibited a correspondingly high level of 6-PGDH enzymatic activity (Submitted). Almost all vanadium ions are reduced to V(HI) via V(IV) in vanadocytes (Hirata and Michibata, 1991), although vanadium ions are dissolved in V(V) in sea water. Some reducing agents must, therefore, participate in the accumulation process. V(V) is reported to stimulate oxidation of NAD(P)H; i.e., V(V) is reduced to V(IV) by the addition of NAD(P)H in vitro (Vyskocil etal., 1980; Nour-Eldeen etal., 1985). Taken together, these observations suggest that N A D P H produced in the pentose phosphate pathway conjugates reduction of vanadium from V(V) through V(IV) in vanadocytes of ascidians. Henze (1911, 1912, 1913, 1932) was the first to report that the homogenate of ascidian blood cells is extremely acidic. Almost all subsequent investigations have supported his observation, however, Kustin's group disputed the earlier reports. They reported that the intracellular pH was neutral on the basis of measurements made by a new technique, which involved the transmembrane equilibrium of C-labeled methylamine (Dingley etal., 1982; Agudelo etal., 1983). Hawkins etal (1983) and Brand etal. (1987) also reported nearly neutral values for the pH of the interior of ascidian blood cells, which they determined noninvasively from the chemical shift of P - N M R . However, Frank etal. (1986) demonstrated that the interior of the blood cells ofAscidia ceratodes has a pH of 1.8, basing their results on the new finding that the ESR line width accurately reflects the intracellular pH. Thus, the reported pH inside ascidian blood cells has excited considerable controversy. We consider that the main reason for the extreme variations is that the measurements of pH were made with entire populations of blood cells and not with the subpopulation of vanadocytes specifically. Thus, one or two specific types of blood cells might have a highly acidic solution within their vacuoles, in which vanadium would be present in a reduced state. With this possibility in mind, we designed an experiment in which we combined the separation of each type of blood cell, measurement of pH with a microelectrode under anaerobic conditions to avoid air-oxidation, and ESR spectrometry as a noninvasive method for the measurement of pH to confirm the results obtained with the microelectrode (Michibata etal., 1991a). Three species of vanadium-rich ascidians, Ascidia gemmata, A. ahodori, and A. sydneiensis samea, were used. Blood cells drawn from each species were fractionated by density-gradient centrifiigation, as described above. The distribution of each type of blood cell, the concentrations of protons ([H*]), and the levels of vanadium in each layer of cells are compared in Fig. 2. It is clear that the distribution patterns of protons and vanadium were similar. Thus, the signet ring cells contain high levels of both vanadium and proton in all three species. ESR spectrometry was also used for noninvasive measurements of the intracellular acidity of blood cells (Michibata etal., 1991). The method is based on the ESR line width due to oxo-vanadium [VO(TV)] ions, which increases (Frank etal., 1986) almost linearly from pH 1.4 to pH 2.3. The low pH values obtained with a microelectrode were confirmed not to be artifacts by the fact that the ESR line width also indicated a low pH for the contents of signet ring cellsfromA. 14

31


254 gemmata. To avoid any misunderstandings, we have to note the assumption that the acidic solution was contained in the vacuole of each signet ring cells. However, the greater part of each signet ring cell is, in fact, occupied by the vacuole itself, so the contents of the vacuole are almost equivalent to the contents of the cell. A comparison of pH values with the levels of vanadium in the signet ring cells of three different species, as shown in Table 3, suggested that there might be a close correlation between a higher level of vanadium and lower pH, namely, a higher concentration of protons. It is well known that H -ATPases can generate a proton-motive force by hydrolyzing ATP. Therefore, we examined the presence of H -ATPase in the signet ring cells of the ascidian Ascidia sydneiensis samea (Uyama etal., 1994). The vacuolar-type H -ATPase is composed of several subunits, and subunits of 72 kDa and 57 kDa have been reported to be common to all eukaryotes examined. Antibodies prepared against the 72 kDa and 57 kDa subunits of a vacuolar-type H -ATPase from bovine chromaffin granules did indeed react with the vacuolar membranes of signet ring cells. Immunoblotting analysis confirmed that the antibodies reacted with specific antigens in ascidian blood cells. Furthermore, addition of bafilomycin A a specific inhibitor of vacuolar-type H ATPases (Bowman etal, 1988), inhibited the pumping function of the vacuoles of signet ring cells, resulting in neutralization of the contents of the vacuoles. We are trying to obtain direct evidence for such an association. The route for the accumulation of vanadium ions from seawater in the blood system has not yet been revealed. The uptake of vanadium ions was studied with radioactive vanadium ions (^V). Previous studies were, however, with afew exceptions (Hawkins etal, 1980; Romania/., 1988) commonly designed with an assumption of the direct uptake of vanadium ions from the surrounding seawater and were, therefore, limited in their determination of how much vanadium was incorporated directly into some tissues (Goldberg etal., 1951; Bielig etal, 1963; Dingley etal, 1981; Michibata etal., 1991b). However, generally, heavy metal ions incorporated into the tissues of living organisms are known to bind to macromolecules such as proteins. Using an anion-exchange column, we first succeeded in isolating a vanadium-associated protein (VAP) composed of 12.5 kDa and 15 kDa peptides with a minor peptide of 16 kDa (Kanda et al., 1997). We raised a monoclonal antibody against V A P , designated F8DH. Immunoblot analysis showed that F8DH recognized 2 related peptides of 15 kDa and 16 kDa of V A P (Wuchiyama etal, 1997). Using F8DH, V A P was shown to be in the cytoplasm of vanadocytes and compartment cells, both of which were reported to contain vanadium. F8DH also stained the vanadocytes distributed in the connective tissues around the alimentary canal, suggesting that vanadocytes in the connective tissue contained VAP. Furthermore, blood cells of 3 different species of ascidian having high levels of vanadium, A. sydneiensis samea, A. ahodori, and Ciona intestinalis, showed reactivity of F8DH but little reactivity was observed in 2 species having less vanadium, Halocynthia roretzi snaPyura michaelseni, suggesting that V A P recognized by F8DH is a common protein in vanadium-rich ascidians. Biochemical characterization of V A P and cloning of the gene encoded V A P are in progress. +

+


+

+

+

1 ?


255

3,000 Ε

10"

2

Ο

100


~ 2,000|

η

Ε

ο

C

-

V 1,000| ο

Layer

I J L

1 234 MC

1 234 1234 SRC CC

ί

1 234 [H*J

c 50

Ci)

Γ

"Ε

Ξ

δ

1

The Accumulation Mechanism of Vanadium by Ascidians - ACS

Recommend Documents