Abstract
Effects of calpain-I purified from human erythrocytes on phosphatidylinositol hydrolysis activity of phospholipase C (PI-PLC) were investigated by using human platelet lysate and the partial purified enzymes. PI-PLC activity in human platelet lysate prepared in the absence of thiolprotease inhibitors (leupeptin and EST) was higher than that prepared in the presence of inhibitors. This result implied that PI-PLC was activated by thiolprotease in platelets during preparation of platelet lysate. When the thiolprotease inhibitors were removed by gel filtration from platelet lysate prepared in the presence of the inhibitors and calpain-I was added into the preparation, the PI-PLC activity was enhanced in correlation with the amount of calpain-I. PI-PLC in platelets lysate was partially purified by phenyl-Sepharose and Ultrogel AcA22 column chromatography. PI-PLC was separated to two isozymes by phenyl-Sepharose chromatography. The apparent molecular weight of these isozymes was about 200kDa in AcA22 gel filtrations. One of the PI-PLC, which was adsorbed with phenyl-Sepharose column and eluted with the buffer containing 50% ethyleneglycol, was not influenced by the preincubation with calpain-I, but the activity of the other one, which was not adsorbed with phenyl-Sepharose column, was enhanced three times by the preincubation with calpain-I. These results indicate that two isozymes of PI-PLC are located in platelets and one of the enzymes is activated by calpain-I.
