Preparation of blood cells

Specimens of the vanadium-rich ascidian, Ascidia sydneiensis samea, were collected at the Otsuchi Marine Research Center, Ocean Research Institute of the University of Tokyo, Otsuchi, Iwate, Japan and at the Ushimado Marine Biological Station of Okayama University, Ushimado, Okayama, Japan. They were maintained in an aquarium that contained circulating natural seawater at each laboratory at 18°C until use.

After the tunic was removed, blood was drawn by puncturing the heart and pooled in 20 volumes of ice-cold buffer solution (buffer A) that contained 400 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, and 50 mM Tris-HCl (tris-hydroxymethyl aminomethane) at pH 8.5. The pooled blood was centrifuged at 400 × g for 10 min at 4°C. The precipitated blood cells were composed of two layers of cells: The upper layer, dominantly consisting of a subpopulation of giant cells, was discarded. The lower one, consisting of the other types of blood cells including vanadocytes, was resuspended in buffer A and again centrifuged at 400 × g for 10 min at 4°C. The pelleted cells were suspended in the other buffer solution (buffer B) that contained 1 mM DTT (dithiothreitol), 1 mM PMSF (phenylmethylsulfonyl-fluoride) and 10% (v/v) glycerol in buffer A. The suspended sample was rapidly frozen in liquid nitrogen and then kept in a deep freezer at −85°C until use.

Isolation of vanadium-associated protein (VAP)

After the frozen blood cells were thawed on ice, they were centrifuged at 400 × g for 10 min at 4°C to remove the cell debris. The supernatant was centrifuged at 100,000 × g for 60 min at 4°C. The supernatant obtained was dialyzed against a 50 mM Tris-HCl buffer containing 1 mM PMSF, 20 μg/ml of pepstatin A, 10 μg/ml of leupeptin and 10 μg/ml of chymostatin at pH 7.4 for 24 hr at 10°C in order to remove the inorganic vanadium ions which did not associate with the proteins. The dialyzed sample was centrifuged at 100,000 × g for 60 min at 4°C, and the supernatant was applied to a DEAE-Sephacel anion-exchange column (ø 1.0 × 10 cm length) which had been equilibrated with a 50 mM Tris-HCl buffer at pH 7.4. After the column was washed with the same buffer solution at 0.5 ml/min and the non-adsorbed substances were eluted, proteins were eluted in 3.4 ml-fractions from the column with increasing NaCl solution, step-wise from 0 to 400 mM in the buffer solution. The concentrations of the protein and vanadium in each fraction were determined by the method of Bradford (1976) using BSA (bovine serum albumin) as the standard and by an atomic absorption spectrometry (Michibata et al., 1990), respectively.

SDS-PAGE

The SDS-PAGE sample was dissolved in a sample dissociation buffered solution containing 62.5 mM Tris-HCl, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol and 2.3% (w/v) SDS at pH 6.8. It was subjected to electrophoresis on a 14% uniformed SDS-PAGE for 12 hr with a 10 mA constant current. Coomassie Brilliant Blue R-250 was used to stain the proteins.

Preparing antibodies

The anti-VAP antibody was prepared by the immunization of an albino rabbit. A 3.5 ml-aliquot of peak 1 fractions that contained vanadium-associated proteins (ca. 100 μg protein/3.5 ml) after elution from a DEAE-Sephacel column was dialyzed against a 5 mM NaCl solution for 6 hr at 4°C. The sample obtained was then lyophilized; it was dissolved in 0.7 ml of physiological saline and mixed with 0.7 ml of Freund's complete adjuvant. The mixture was hypodermically injected into a rabbit, and the injection was repeated two weeks after the first injection to boost the titer of the antibody. Three days after the last injection, blood was collected from the ear. The blood was used for the preparation of the anti-VAP serum, which was allowed to stand at 37°C for 1 hr, and was then incubated at 4°C overnight. The serum was removed from the clot, and any remaining insoluble material was removed by centrifugation at 10,000 × g for 10 min at 4°C. After the preparation, the serum was stored at −30°C until use.

The anti-VAP antibody was purified as follows: Peak 1 fractions containing about 500 μg protein were subjected to SDS-PAGE. The proteins separated on SDS-PAGE were electrophoretically blotted onto nitrocellulose paper and sliced into several bands which corresponded to the VAP. The paper sliced was then washed with Tris-buffered saline (TBS), which consisted of 140 mM NaCl and 10 mM Tris-HCl buffer at pH 7.2, for 20 min and soaked in TBS containing 1% (w/v) BSA for 1 hr. The paper was then exposed to the anti-VAP serum, which was diluted 20-fold with TBS containing 1% BSA, for 2 hr at room temperature. After washing three times with TBS containing 0.01% (v/v) Tween 20 for 10 min, the paper was incubated in a 0.1 M glycine-HCl buffer (pH 3.0) and vigorously agitated in order to release the antibodies from the antigen. The solution was immediately neutralized with 1 M Tris-HCl (pH 9.0) containing 100 μg BSA. The solution was then dialyzed against 10 mM NH₄HCO₃ for 3 hr at 4°C and concentrated with Centricon 10 (Amicon, Inc., Mass. USA) to use as purified antibodies (purified anti-VAP).

Immunoblot analysis

Homogenates of blood cells containing 40 μg protein and peak 1 fractions containing 5 μg protein were dissolved in a sample dissociation buffered solution and subjected to SDS-PAGE. For immunoblot analysis, each sample subjected to SDS-PAGE was transferred to nitrocellulose paper. The paper was then soaked in TEN buffer, which consisted of 150 mM NaCl, 1 mM EDTA, 25 mM Tris-HCl buffer at pH 7.4 containing 1% BSA, for 3 hr and exposed to purified anti-VAP antibody, which was diluted at a ratio of 1 to 500 in TEN buffer containing 1% BSA, for 1 hr. The nitrocellulose paper was twice washed with TEN buffer containing 0.01% Tween 20 for 30 min and incubated with the anti-rabbit IgG (H + L)-HRP conjugate (Organon Teknika Corporation, Philadelphia, USA), which was diluted at a ratio of 1 to 2,500 with TEN buffer containing 0.01% Tween 20, for 30 min. After washing six times with the TEN buffer containing 0.01% Tween 20 for 15 min, the nitrocellulose paper was incubated with ECL Western blotting detection reagents (Amersham International, Plc., Buckinghamshire, England). Finally, the nitrocellulose blot was exposed to Hyperfilm-ECL (Amersham International, Plc.).

Hypotonic, LiCl and digitonin treatments

To examine whether or not vanadium ions are associated with the membranes of blood cells and whether or not the VAP is derived from the cytoplasm of blood cells, the blood cells were treated with hypotonic, LiCl and digitonin solutions, respectively. The hypotonic treatment was carried out as follows: An aliquot of 200 mg of the pelleted blood cells was suspended in 40 μl of hypotonic solution (1 mM Tris-HCl at pH 7.4 containing 10 μg/ml PMSF, 10 μg/ml leupeptin and 10 μg/ml chymostatin) for 30 min at 4°C and then centrifuged at 100,000 × g for 60 min at 4°C. The total amount of the supernatant obtained was mixed with 10 μl of a sample dissociation buffer solution containing 25 mM Tris, 125 mM glycine, 5% (v/v) β-mercaptoethanol, 2.4% (w/v) SDS and 20% (v/v) glycerol and subjected to SDS-PAGE. An aliquot of the blood cells which had undergone the hypotonic treatment was further treated with LiCl as follows: The total amount of pelleted sample after treatment with hypotonic solution were further treated with LiCl. The sample was mixed with 1 ml of LiCl solution (1 mM Tris-HCl and 1 M LiCl at pH 7.4) and centrifuged at 100,000 × g for 60 min at 4°C. The supernatant obtained was mixed with a sample dissociation buffer solution and subjected to SDS-PAGE as same as the hypotonic treated sample. For the treatment with digitonin, an aliquot of 200 mg of the pelleted blood cells was resuspended in 1 ml of 1 mM Tris-HCl at pH 7.4 containing 0.5% digitonin, and the subsequent treatments were carried out in the same manner as described above. As the control, isotonic treament was done as follows; an aliquot of 200 mg of the pelleted blood cells was suspended in 40 μl of isotonic solution contained 460 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, and 50 mM Tris-HCl at pH 8.5 and the subsequent treatments were done in the same manner as described above.

Isolation of vanadium-associated proteins (VAP)

Approximately 50% of the vanadium contained in the initial precipitation of the blood cells was lost after the treatment of freezing and thawing, and approximately 90% of the initial vanadium was further lost from the sample after the subsequent dialysis, as shown in Table 1. When the dialyzed sample was loaded onto a DEAE-Sephacel anion-exchange column, one major peak designated as peak 1, containing both proteins and vanadium, was obtained in fractions 3–9, and the other major peak designated as peak 2, containing only vanadium, was found in fractions 30–55 followed by gradients of 200 mM to 400 mM NaCl, as shown in Fig. 1. Peak 1 fractions contained 25 μg/ml of proteins and 0.95 μg /ml of vanadium. On the other hand, to confirm that the vanadium in peak 2 fractions was an inorganic chemical species, an inorganic solution of 6 μM vanadate (Na₃VO₄) was applied to the column under the same conditions. Consequently, the elution pattern of peak 2 was essentially indistinguishable from that of the inorganic solution (data not shown).

Table 1

Contents of vanadium and proteins during isolation of the vanadium-associated proteins

Fig. 1

Isolation of a vanadium-associated protein (VAP) from the blood cells of the vanadium-rich ascidian, Ascidia sydneiensis samea. When the supernatant of the blood cells was applied to a DEAE-Sephacel anion-exchange column, one major peak designated as peak 1, containing both proteins and vanadium, was obtained in fractions 3–9, and the other major peak designated as peak 2, containing only vanadium, was found in fractions 30–55 followed by gradients of 200 mM to 400 mM NaCl. Peak 1 fractions contained 25 μg/ml of proteins and 0.95 μg/ml of vanadium. Peak 2 fractions contained an inorganic vanadium species. The insertion is SDS-PAGE indicating that peak 1 contained at least two types of peptides whose molecular weights were estimated to be 12.5 kDa and 15 kDa, respectively.

SDS-PAGE and immunoblot analysis

SDS-PAGE revealed that peak 1 contained at least two types of peptides whose molecular weights were estimated to be 12.5 kDa and 15 kDa, respectively, as shown in Figs. 1 (insertion) and 2A. The comparison of the SDS-PAGE patterns of peak 1 fractions with those of the homogenate of the blood cells, supernatant after freezing and thawing, and the dialyzed sample shows that the treatment of freezing and thawing removed the higher molecules of proteins and resulted in the desirable purification of the vanadium-associated protein. Purification of the vanadium-associated protein was carried out in the same manner but without Ca²⁺ to examine the effects of Ca²⁺-dependent proteases on extraction of peptides from ascidian blood cells, consequently, several bands more than 45 kDa appeared on SDS-PAGE but little effects were observed around 15 kDa (data not shown).

Fig. 2

SDS-PAGE and immunoblot analysis. SDS-PAGE (A) revealed that peak 1 contained at least two types of peptides whose molecular weights were estimated to be 12.5 kDa and 15 kDa, respectively. The comparison of the SDS-PAGE patterns of peak 1 fractions with those of the homogenate of the blood cells, supernatant after freezing and thawing, and the dialyzed sample shows that the treatment of freezing and thawing removed the higher molecules of proteins and resulted in the desirable purification of the vanadium-associated protein. Immunoblot analysis (B) shows that a polyclonal antibody, anti-VAP, recognized at least two main peptides of 12.5 kDa and 15 kDa with a minor peptide of 16 kDa in the homogenate of blood cells and in peak 1 fractions.

When the blood cells were treated with a hypotonic solution and digitonin, around 15 kDa bands were stained much more deeply than those in the control (Fig. 3), which indicates that the 15 kDa proteins contained in the cytoplasm of blood cells were released by the treatments, namely, VAP existed intrinsically in the cytoplasm of the blood cells. The treatment with LiCl also released some amounts of proteins, suggesting that the VAP might bind with cell membranes by ionic interaction.

Fig. 3

Treatment with hypotonic solution, digitonin, and LiCl. The blood cells treated with hypotonic solution, digitonin, or LiCl were submitted for SDS-PAGE. Around 15 kDa bands were stained much more deeply than those in the control by the treatment with hypotonic solution or with digitonin, indicating that VAP existed intrinsically in the cytoplasm of the blood cells. The treatment with LiCl also released some amounts of proteins, suggesting that the VAP might bind with cell membranes by ionic interaction.

Immunoblot analysis confirmed that at least two main peptides of 12.5 kDa and 15 kDa with a minor peptide of 16 kDa contained in peak 1 fractions were recognized by a polyclonal antibody, anti-VAP (Fig. 2).