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Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity
M Bharadwaj1, D Bharadwaj, R N Hati
1Department of Physiology, Indian Institute of Chemical Biology, Calcutta, India.
Abstract:
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.
Insights
Researchers purified a novel membrane protease with thrombin-like activity from rat mitochondria. This serine protease exhibits plasma-coagulating and fibrinogen-clotting functions, distinct from other known proteases.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Mitochondria contain various enzymes, but their specific roles in coagulation pathways are not fully elucidated.
- Thrombin-like enzymes play crucial roles in hemostasis and thrombosis.
- Understanding novel proteases is key to deciphering complex biological processes.
Purpose of the Study:
- To purify and characterize a novel membrane protease from rat submaxillary gland mitochondria.
- To investigate the enzyme's enzymatic activity, substrate specificity, and potential role in blood coagulation.
- To differentiate this enzyme from other known serine proteases.
Main Methods:
- Purification of the enzyme to homogeneity using standard biochemical techniques.
- Enzyme characterization including molecular mass determination (SDS/PAGE, gel filtration), isoelectric point, and optimal pH.
- Enzyme activity assays using synthetic substrates and purified fibrinogen.
- Inhibition studies with various protease inhibitors.
- High-Performance Liquid Chromatography (HPLC) analysis for fibrinopeptide release.
Main Results:
- A 45 kDa glycoprotein serine protease with thrombin-like activity was isolated.
- The enzyme demonstrated optimal activity at pH 10.5 and showed specificity for arginine at the P1 position.
- Significant plasma-coagulating and fibrinogen-clotting activities were observed.
- Inhibition studies confirmed its serine protease nature and identified aprotinin as a potent inhibitor.
- HPLC analysis confirmed the release of fibrinopeptides A and B.
Conclusions:
- A novel mitochondrial membrane protease with thrombin-like activity has been identified and characterized.
- This enzyme plays a role in plasma coagulation and fibrinogen clotting, distinct from trypsin and chymotrypsin.
- The findings contribute to understanding mitochondrial function and protease diversity in biological systems.