Abstract
Sea squirts accumulate vanadium compounds with potent antidiabetic activity, which are involved in immune defense. In this study, vanadium concentrations of fresh blood plasma, intestine, and muscle of the sea squirt Halocynthia roretzi were 6.3, 3.7 and 2.1 mg/kg respectively. Two vanadium binding proteins (VBPs) from blood plasma and intestine were purified through (NH(4))(2)SO(4) precipitation, and DEAE-Sepharose ion exchange and Sephacryl S-200 HR gel filtration chromatography, in that order. The purity and yield of the intestine and blood plasma vanadium binding proteins, VBP(intestine) and VBP(blood plasma,) were 13.4 folds and 7.1%, and 20.9 folds and 6.8%, respectively. There were two protein bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 24.3 and 68.8 kDa and one with 96.7 kDa on the native-PAGE of VBP(blood plasma), whereas only one protein band of VBP(intestine) on the SDS-PAGE with 26.5 kDa. Antioxidant activities of VBPs were lower than that of ascorbic acid. Both VBPs exerted strong inhibitory activity against Saccharomyces cerevisiae and mild against Bacillus stearothermophilus and rat intestinal α-glucosidase. IC(50) values of VBP(intestine) and VBP(blood plasma) against S. cerevisiae α-glucosidase were 28.34 and 12.60 μg/ml, respectively. The K(m) , V(max) , k(cat) , and k(cat)/K(m) values of VBP(intestine) and VBP(blood plasma) were 4.29, 0.036, 6.58 and 1.53 × 10(3), and 7.63 mM, 0.057 mM/min, 10.41 s(-1) and 1.36 × 10(3) (M sec)(-1), respectively. There was a synergistic interaction between VBP(blood plasma) and VBP(intestine) on rat intestinal α-glucosidase inhibitory activity.