Changes in inhibitory effect of vanadate on the plasma membrane H+-ATPase under trypsin treatment

The changes in inhibitory effect of vanadate on the plasma membrane H+-ATPase were studied with mild trypsin treatment using plasma membrane vesicles purified from soybean (Glycine max L.) hypocotyles by sucrose gradient centrifugation. Results showed that under mild trypsin treatment the ATPase ATP hydrolysis activity was increased significantly. It was also found that the inhibitory effect of vandate was reduced after proteolysis. In the presence of 2 mmol/L vanadate, the ATP hydrolysis activity of the cleaved ATPase was inhibited by only 53.49%, while that of the un-cleaved ATPase was inhibited by 64.13%. Kinetic studies indicated that both the K-m values and the inhibition type of vanadate were not affected by trypsin treatment. Upon proteolysis, K-m remained as 0.34 mmol/L, while vanadate was still an uncompetitive inhibitor. Taking together, the structure and activity of the ATPase phosphatase domain were affected by trypsin treatment, implying that this domain might be regulated by the C-terminal end of the plasma membrane H+-ATPase.