Renal dipeptidase: Localization and inhibition

Abstract

The principle locus of porcine renal dipeptidase activity has been found to be the microsomal fraction of kidney cortex. When the microsomal preparation was incubated with the enzymes, chymotrypsin, trypsin, lipase, and ribonuclease, the peptidase was released to no greater degree than other protein components of this fraction. Treatment of the microsomal fraction with deoxycholate and sodium dodecyl sulfate led to the identification of the dipeptidase within the resulting ribosomal fraction. Relatively large-scale isolation methods applied to the preparation of renal dipeptidase resulted in an enzyme of purity comparable to that reported in previous studies. The molecular weight of the purified peptidase was estimated by gel filtration on Sephadex G-200 to be approximately 90 000. The enzyme was shown to be competitively inhibited by phosphate and phosphate esters, and the inhibition demonstrated to be instantaneous and completely reversible upon dilution. A comparison of K i values for the adenine series indicated that the effectiveness of inhibition was in the order ATP > ADP > AMP > inorganic phosphate. It is suggested that the phosphate inhibition occurs as a result of the formation of a ternary (peptidase-Zn-inhibitor) complex at the active site of the enzyme. Inhibition of renal dipeptidase by 1,10-phenanthroline was measurably time dependent and could not be completely reversed by dilution or by the addition of excess zinc ions. However, removal of chelating agent followed by dialysis against zinc containing buffers led to reversal of inhibition. It seems likely that the inhibitory effect of 1,10-phenanthroline is caused by the removal of zinc from the active site of the peptidase to produce an inactive apoenzyme. The native enzyme can be reconstituted with additional zinc to restore full activity. When the rate of peptidase-catalyzed hydrolysis of dipeptides was measured in the presence of the free amino acid components, inhibition of hydrolysis was clearly demonstrated. The amino acids with bulkier side chains were the more effective inhibitors regardless of their positions as N-terminal or C-terminal components of the substrate. There was observed a lack of esterase activity against amino acid and peptidyl esters indicating that the free carboxyl group is required for renal dipeptidase activity.