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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-.

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The effect of different chemical reagents on renin has been investigated. After treatment of hog renin with acetic anhydride, 1-fluoro-2,4-dinitrobenzene, diazobenzene sulfonic acid and phenyl isocyanate, the enzymic activity assayed both biologically and chemically was depressed, whereas no effect occurred after treatment with diisopropyl fluorophosphate, p-chloromercuribenzoate and after dialysis against EDTA. Human renin, too, is inactivated by acetylation, diazo coupling and dinitrobenzoylation. The results indicate that one or several amino groups of the renin molecule are essential for enzymic activity.

Volumem 49, Fasciculus 6 (1966) -No. 225 1903 225. Un substrat pour le dosage chimique de la rknine par M. Roth et Anja Reinharz (14 VI 66)

The milk-ejecting response of lactating mouse mammary gland tissue to ovine pineal extracts indicated the presence of a neurohormone-like bioactivity in this tissue. After successive fractionation on gel permeation chromatography and reversed-phase liquid chromatography (HPLC) in conjunction with radioimmunoassays (RIA), it was demonstrated that the milk-ejection response to ovine pineal components with an Mr less than 1,000 corresponded to a biologically active peptide sequence that probably differs from that of arginine vasopressin, arginine vasotocin, and oxytocin and from peptides with a COOH-terminal Pro-Arg-Gly-amide ending. Gel permeation chromatography in formic acid appeared also to indicate the presence of a noncovalent interaction of the neurohormone-like bioactivity with proteins (Mr greater than 25,000) of the pineal.

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