Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess 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 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.
Bharadwaj
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M
Department of Physiology, Indian Institute of Chemical Biology, Calcutta, India.
Bharadwaj
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Hati
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D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D002417:Cattle; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000201:enzymology*; D007526:Isoelectric Point; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats; D013363:Submandibular Gland / Q000201:enzymology* / Q000648:ultrastructure; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology
D008565:Membrane Proteins; D005340:Fibrinogen; D014360:Trypsin Inhibitor, Kunitz Soybean; D010450:Endopeptidases; D013917:Thrombin