Renal Aminopeptidase- and Copper-activated Peptide Hydrolysis.

Robinson, Birnbaum, and Greenstein (1) in 1953 accomplished the isolation of renal aminopeptidase from fresh hog kidney by extracting the activity from the particulate fraction of kidney homogenate with I-butanol and thereafter fractionating between 50 and 75% saturation with ammonium sulfate at pH 7. About 30% of the final product was extractable by organic solvents, the extractable material appearing to be lipid in nature. These workers demonstrated inactivation of renal aminopeptidase with cyanide and sulfhydryl compounds and with both saturated and unsaturated peptides as substrates. They further showed that the initial rates of the enzymic hydrolysis could be increased by addition of zinc or cobalt ions. Earlier work by Yudkin and Fruton (2) had resulted in the preparation of renal aminopeptidase from desiccated and defatted swine kidney powder by extraction with 2% NaCl solution and isolation of the activity as an ammonium sulfate fraction between 40 and 80% saturation. With this preparation it was possible to reduce markedly the activity against glycyldehydrophenylalanine by dialysis for 4 days at 5” against demineralized water. Of the various metals tested, only Zn+f was found to reactivate the enzyme completely; CoClp, MnSO+ and MgS04 were without appreciable effect. Campbell, Yudkin, and Klotz (3), using aminopeptidase prepared in the manner described by Yudkin and Fruton (Z), demonstrated complete inhibition of activity by the sulfhydryl compounds, 0.002 M cysteine or 0.002 M thioglycolate. The removal of sulfhydryl inhibition was effected by dialysis or by the addition of 0.001 M ZnCln. In the present investigation an ion exchange chromatographic technique was employed to further purify renal aminopeptidase. The use of the unsaturated peptide, glycyldehydrophenylalanine, as the substrate for aminopeptidase in the isolation procedure provided a facile assay making possible the analysis of a large number of fractions in a relatively short period of time. Furthermore, the spectrophotometric assay provided a means of obtaining reliable quantitative data for kinetic interpretation. In a preliminary report Campbell et al. (4) indicated that the hydrolysis of glycyldehydrophenylalanine could be induced by cupric ions. In this paper a study of copper-induced hydrolysis is presented as a model system for comparison with the enzymecatalyzed reaction.