Studies on new intracellular proteases in various organs of rat. 1. Purification and comparison of their properties.
1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002852:Chromatography, Ion Exchange; D007413:Intestinal Mucosa / Q000276:immunology; D007421:Intestine, Small / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009928:Organ Specificity; D010447:Peptide Hydrolases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011733:Pyridoxamine / Q000494:pharmacology; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
D000596:Amino Acids; D002413:Cations, Divalent; D002414:Cations, Monovalent; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D010447:Peptide Hydrolases