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 reducing 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 fluorophosphate, benzamidine, aprotinin and antipain suggested the enzyme to be a serine protease. Other inhibitors such as EDTA, soya-bean trypsin inhibitor, lima-bean trypsin inhibitor, TosLysCH2Cl and chymostatin did not alter the activity. The enzyme showed affinity towards different synthetic substrates (p-nitroanilide derivatives) containing arginine at the P1 position. Kinetic studies revealed that kcat./Km was highest with the substrate N-Bz-Phe-Val-Arg-p-nitroanilide. The enzyme exhibits significant plasma-coagulating activity. The coagulation initiated by the enzyme was not altered by concanavalin A, indicating that the carbohydrate moiety of the enzyme is not essential for this reaction. 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-clotting activity of the enzyme. Fibrinogen coagulation by the enzyme was blocked in the presence of aprotinin, a protease inhibitor. Release of fibrinopeptides A and B from bovine fibrinogen by the enzyme has been shown by HPLC analysis. Our studies reveal that the enzyme reported here differs from trypsin, chymotrypsin and other mitochondrial proteases reported so far.
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January 1996
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Research Article|
January 01 1996
Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity
Mausumi BHARADWAJ;
Mausumi BHARADWAJ
1Department of Physiology, Indian Institute of Chemical Biology, 4-Raja S.C. Mullick Road, Jadavpur, Calcutta-700 032, India
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Dwaipayan BHARADWAJ;
Dwaipayan BHARADWAJ
1Department of Physiology, Indian Institute of Chemical Biology, 4-Raja S.C. Mullick Road, Jadavpur, Calcutta-700 032, India
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Ratha N. HATI
Ratha N. HATI
*
*To whom correspondence should be addressed.
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Biochem J (1996) 313 (1): 193–199.
Article history
Received:
August 07 1995
Accepted:
August 21 1995
Citation
Mausumi BHARADWAJ, Dwaipayan BHARADWAJ, Ratha N. HATI; Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity. Biochem J 1 January 1996; 313 (1): 193–199. doi: https://doi.org/10.1042/bj3130193
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