A new blood-coagulating protease in mitochondrial membranes of rat submaxillary glands. Purification and characterization of protease and its blood-coagulating activity.
An integral membrane protease was solubilized and purified to homogeneity from rat submaxillary mitochondria. The purified enzyme could coagulate rabbit plasma. The molecular mass of the enzyme is 22 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions and 24 kDa on gel filtration on a Sephadex G-100 column. Its isoelectric point is 4.2-4.25. Enzyme activity is strongly inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, benzamidine, aprotinin, and antipain, suggesting the enzyme as a serine protease. Its pH optimum for activity is 8.5. Zn2+ is strongly inhibitory; at 1 mM concentration it produced 72% inhibition. The enzyme is active toward different synthetic substrates (p-nitroanilide derivatives) containing Arg at the P1 position with blocked NH2 terminus. Kcat/Km was highest with the substrate N-Bz-Pro-Arg-pNa (where Bz is benzoyl and pNA is paranitroanilide). The purified enzyme coagulates rabbit plasma in a dose-dependent manner. Plasma coagulation by the enzyme is completely blocked in the presence of aprotinin or soybean trypsin inhibitor, suggesting that protease activity is required for this coagulation reaction. Antibody raised against the purified enzyme inhibits the plasma coagulation initiated by the enzyme. The enzyme can correct the prolonged clotting time of factor X-deficient human plasma but is unable to convert purified fibrinogen to fibrin clots, indicating factor Xa-like activity of the enzyme. The enzyme has the ability to activate prothrombin. Several properties of the enzyme distinguish it from other reported submaxillary proteases.
Bharadwaj
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D
Indian Institute of Chemical Biology, Jadavpur, Calcutta, West Bengal.
Roy
M S
MS
Bose
D
D
Hati
R N
RN
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000201:enzymology*; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011516:Prothrombin / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D013363:Submandibular Gland / Q000201:enzymology*; D013379:Substrate Specificity
D011480:Protease Inhibitors; D011516:Prothrombin; D010450:Endopeptidases; D012697:Serine Endopeptidases