Studies on Renin with Synthetic Substrates

Abstract

Renin has been shown to catalyze the hydrolysis of the protected octapeptide, Z-Pro-PheHis-Leu-Leu-Val-Tyr-Ser-B-naphthylamide, a t the leucyl-leucyl bond. When the octapeptide substrate is incubated with renin and an excess of the auxiliary enzyme, aminopeptidase M, the fluorescent B-naphthylamine is liberated a t a rate related to renin concentration. A chemical assay of renin based on these facts is described in detail. When assayed with this method, kidney renin from man and pig shows pH optima of 5.4 and 5.7 respectively. Renin from submaxillary glands of mice, which also attacks the substrate, is optimally active a t pH 5.4.The limit of sensitivity of the assay is 0.001 Goldblatt units for human renin and 0.01 Goldblatt units for pig renin. Trials with other possible synthetic substrates showed that Z-Pro-PheHis-Leu-Leu-,!?-naphthylamide is poorly hydrolyzed by renin, and Z-Leu-Leu-B-naphthylamide not at all.Action of renin on a substrate of known structure was first reported by Skeggs and collaborators [l], who showed that pig renin is capable of hydrolyzing the leucyll0-leucylll bond of the tetradecapeptide A fluorigenic substrate of renin has been synthesized in our laboratory [5]. This substance, Z-ProPhe-His-Leu-Leu-Val-Tyr-Ser-B-naphthylamide, can be used to determine the enzymatic activity of purified renin by a chemical method which is simpler and affords a better reproducibility than the currently available biological methods of renin assay.The assay is based on the fact that p-naphthylamine is highly fluorescent, whereas its N-acyl derivatives are not. Renin splits the substrate in two moieties, one of which is the non-fluorescent peptide derivative Leu-Val-Tyr-Ser-B-naphthylamide. The four amino acids it contains are then successively split off by the auxiliary enzyme, aminopeptidase M [6], added in excess. Free P-naphthylamine, easily measurable by fluorimetry, is thus liberated a t a rate related to the renin activity. Since it requires a free terminal amino group for activity, aminopeptidase M is without action on the octapeptide.Unusual Abbreviation. Benzyloxycarbonyl-, Z-.

Purification of renin and prorenin.

“…Roth and Reinharz 18 had synthesized a fluorogenic substrate for renin and a simple method for fluorometric determination of renin activity was devised. 19 This method was to become the critical factor in the purification of renin. On my request, they were kind enough to send us a generous supply of their fluorogenic substrate with the following structure:…”

Studies on Pituitary Catkepsin D with Artificial Substrates

“…Previously published procedures were utilized for the synthesis of Z-Pro-Phe-HisLeu-Leu-Val-Tyr-Ser-ß-naphthylamide and Z-Val-Tyr-Ser-ß-naphthylamide [1], and of Z-Leu-Leu-ß-naphthylamide, Z-Leu-Val-Tyr-Ser-ß-naphthylamide and ZPro-Phe-His-Leu-ß-naphthylamide [2],…”

“…In previous publications [1,2] we reported the synthesis and use of an artificial octapeptide substrate of renin (EC 3. 4.…”

Aldosterone and renin measurements

“…The ®rst use of a synthetic¯uorogenic substrate was described in 1969. 53 The¯uorescent compound produced in the reaction, b-naphthylamine, is carcinogenic and the method was never fully developed. Several years later two new¯uorogenic peptides were developed which contained 4-methylcoumaryl-7-amide.…”