Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity

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.

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti

“…Inhibition of the peptidolytic activity of both de- and glycosylated forms of TrCB2 was tested at pH optimum 6.0 with 100 μM Z-Arg-Arg-AMC, a substrate specific for cathepsin B and a spectrum of differentiating peptidase inhibitors at 50 μM concentrations: E-64 [ N - trans -(epoxysuccinyl)- l -leucine 4-guanidinobutylamide], irreversible broad spectrum Clan CA cysteine peptidase inhibitor; CA-074 [ N -( l -3- trans -propylcarbamoyloxirane-2-carbonyl)-Ile-Pro-OH], irreversible, selective, of cathepsin B ( Towatari et al, 1991 ); Z-Phe-Ala-CHN 2 [Z-Phe-Ala-diazomethylketone], irreversible, of cathepsins B and L ( Dalton et al, 1997 ); Z-Phe-Phe-CHN 2 [Z-Phe-Phe-diazomethylketone], irreversible, of cathepsins B and L ( Nowak et al, 2004 ); Z-Phe-Tyr-CHN 2 , [Z-Phe-Tyr(tBu)-diazomethylketone], irreversible, of cathepsin L ( Shaw et al, 1993 ); elastatinal [ N -(Na-Carbonyl-Cpd-Gln-Ala-al)-Leu], irreversible, specific, of neutrophil and pancreatic elastases but not other serine peptidases such as trypsin or chymotrypsin ( Bieth, 2004 ); DFP (diisopropylfluoro-phosphate), irreversible, of serine peptidases ( Bharadwaj et al, 1996 ).…”

Proteolytic activity in Microcystis aeruginosa PCC7806 is inhibited by a trypsin-inhibitory cyanobacterial peptide with a partial structure of microviridin

“…Under the same assay conditions, the thrombin-specific inhibitor, anti-thrombin III led to a minor inhibition (15%), while carboxypeptidase (metal protease)-specific inhibitors 1,10-phenanthroline and EDTA, and cysteine protease-specific inhibitors E64 and iodoacetamide were without any significant effect (p<0.001; not shown). The concentrations of the inhibitors selected have previously shown to inhibit the corresponding animal proteases by over 90% (Bharadwaj et al 1996;Michaud 1998).…”