ELECTROPHORETIC EVIDENCE FOR ISOZYMES OF ARYLAMIDASE ("AMINOPEPTIDASE") IN THE RAT. I. RENAL,SERUM AND URINARY ENZYMES SPLITTING dl -ALANYL-2-NAPHTHYLAMIDE AND l -LEUCYL-2-NAPHTHYLAMIDE

Abstract

Bilateral nephrectomy elicited no changes of serum blood levels of arylamidase assayed with two chromogenic substrates (l-leucyl-2-naphthylamide and dl-alanyl-2-naphthylamide) two days after the operation. Since rat urine contains similar enzymes it was postulated that the urinary enzyme is of renal origin and distinct from serum arylamidase. For this purpose, electrophoretic studies were undertaken. Starch block (for quantitative determinations) and starch gel for zymograms of arylamidase were used. It was shown that both procedures demonstrated the identity of urinary and renal arylamidase, which was distinct from the serum enzyme. The renal and urinary enzyme showed two distinct isozymes: one remaining at the origin and the other migrating somewhat less than the isozyme in serum. It is postulated that the isozyme remaining at the origin corresponds to a membrane-bound form, whereas the electrophoretically-mobile isozyme is found in the cellular supernatant fraction. The arylamidase from serum was represented by a single enzyme which migrated farthest towards the anode.

Histochemical procedures for the demonstration of arylamidase activity in tissues at the light microscopic level permit the localization of enzyme(s) that can hydrolyze synthetic chromogenic naphthylamides containing l-leucyl and dl-alanyl groups (1).

Rat kidneys have a high concentration of histochemically demonstrable arylamidase in the proximal convoluted tubules (2). Blood levels of arylamidase in the normal adult rat vary within a narrow range (3). The observation of arylamidase activity in rat urine raised several questions that led to the studies which are the basis of the present report.

The relationship of serum and urinary arylamidase was the starting point of this investigation. It was speculated that if the urinary enzyme had its origin in the serum, bilateral nephrectomy should alter blood levels of the enzyme. If no such changes occurred, this would suggest that the urinary enzyme was released from the proximal convoluted tubule. In fact, total renal ablation led to no significant variation of serum arylamidase activity.

It was therefore postulated that urinary arylamidase originated in the kidneys and that both are distinct from serum arylamidase. To test this hypothesis, zone electrophoretic studies were undertaken using two different supporting media. It will be shown that distinct molecular forms or isozymes of arylamidase can be separated from rat tissues and fluids by differential electrophoretic mobility. The electrophoretic identity of urinary and renal arylamidase, both of which are distinct from the serum arylamidase, is demonstrated in this study.