pHuman kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme./p
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