PURIFICATION AND PARTIAL CHARACTERIZATION OF THE MAJOR CYSTEINE PROTEASE FROM ENTAMOEBA-INVADENS

Abstract

The purification and partial characterization of a major protease from the parasitic protozoon of reptiles, Entamoeba invadens, is described. The enzyme has a molecular mass of 28 kDa, and three distinct isoelectric points at pH 4.7, 5.7 and 6.3, respectively. As an endopeptidase the enzyme digests denatured protein substrates, such as azocasein with an optimal tunover rate at pH 4.8 with a temperature optimum of 48 degrees C. The protease exhibits exopeptidase activity towards arginine containing dipeptide derivatives. Thus, it splits the chromogenic substrates N-benzyloxycarbonyl-arginine-arginine-4-methoxy-beta-naphthylamide and arginine-arginine-4-methoxy-beta-naphthylamide in the ratio of velocities of 3:1. The kinetic constants for the hydrolysis of N-benzyloxycarbonyl-arginine-arginine-4-methoxy-beta-naphthylamide are: Km 22 mu-M and kcat 172s-1. The enzyme is activated by the thiol reagents cysteine and dithiothreitol and is inhibited by typical cysteine protease inhibitors, such as cystatin, E-64, iodoacetamide and p-chloromercuribenzoate. Although in many of its characteristics it resembles the cathepsin B-like cysteine protease from Entamoeba histolytica, the two enzymes were found to be immunologically different.