CHARACTERIZATION OF A MEMBRANE PROTEASE FROM RAT SUBMAXILLARY-GLAND MITOCHONDRIA THAT POSSESSES THROMBIN-LIKE ACTIVITY

A membrane protease possessing thrombin-like activity was purified to homogeneity from mitochondria of rat submaxillary gland. The molecular mass of the enzyme was determined to be 45 kDa by SDS/PAGE under redu cing conditions and by gel filtration on a Sephadex G-100 column. The enzyme is a glycoprotein and has an isoelectric point of 3.25. Maximum activity was observed at pH 10.5. Inhibition by di-isopropyl fluoroph osphate, benzamidine, aprotinin and antipain suggested the enzyme to b e a serine protease. Other inhibitors such as EDTA, soya-bean trypsin inhibitor, lima-bean trypsin inhibitor, TosLysCH(2)Cl and chymostatin did not alter the activity. The enzyme showed affinity towards differe nt synthetic substrates (p-nitroanilide derivatives) containing argini ne at the P-1 position. Kinetic studies revealed that k(cat.)/K-m was highest with the substrate N-Bz-Phe-Val-Arg-p-nitroanilide. The enzyme exhibits significant plasma-coagulating activity. The coagulation ini tiated by the enzyme was not altered by concanavalin A, indicating tha t the carbohydrate moiety of the enzyme is not essential for this reac tion. Further, this enzyme can catalyse the formation of fibrin clots from purified fibrinogen, which describes its thrombin-like activity. However, an antibody raised against the purified enzyme inhibited the plasma-clotting as well as fibrinogen-clothing activity of the enzyme. Fibrinogen coagulation by the enzyme was blocked in the presence of a protinin, a protease inhibitor. Release of fibrinopeptides A and B fro m bovine fibrinogen by the enzyme has been shown by HPLC analysis. Our , studies reveal-that the enzyme reported here differs-from trypsin, c hymotrypsin and other mitochondrial proteases reported so far.