Abstract
Proline endopeptidase (PEPase) was purified 71800-fold from the soluble fraction of rat liver with a yield of 65.2%. Ammonium sulfate fractionation and chromatographies on diethyl aminoethyl (DEAE)-Sepharose CL-6B, DE-52, blue Sepharose CL-6B, carbobenzoxyglycyl-L-prolyl-AH-Sepharose 4B and Mono-Q were used for the purification of the enzyme. Among the purification procedures, bue Sepharose CL-6B chromatography was the most effective step to eliminate various contaminants. The final enzyme preparation had a high specific activity of 12200 unit/mg protein and showed maximal activity at pH 5.9 toward succinylglycyl-L-proline 4-methylcoumaryl-7-amide (Suc-Gly-Pro-MCA). The Km and Vmax values for Suc-Gly-Pro-MCA were 0.43 mM and 52.0 μmol/min/mg protein, respectively. The apparent molecular weight of rat liver PEPase was estimated to be 68000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 59000 by gel filtration on TSK-Gel G-4000 SW. The enzyme was extremely sensitive to disopropyl fluorophosphate, but no effect was observed with aprotinin, soybean trypsin inhibitor or ethylenediaminetetraacetate. In addition, the enzyme activity was strongly inhibited by p-chloromercuribenzoate and Hg2+. Elastatinal and 1, 10-phenanthroline also significantly inactivated the enzyme. These results indicate that rat liver PEPase was similar to PEPase purified previously from rat brain, porcine liver and other sources in some enzymatic properties and in molecular nature. The distribution of the enzyme was similar to that of the cytosol marker enzyme, lactate dehydrogenase.
