Id. w27 -97 2X/ 7 8 / 0046-000 I $02.00/0 Journal of Clinical Endocrinologyand Metabolism Copyright@ 1978 by the Endocrine Society Comments Vol.46, No. I Printed in U.S.A. Activation of Human fnactive ("Pro-") Renin by Cathepsin D and Pepsin* BRrAN J. MORRTSt Department of Physiology, Uniuersity of California School of Medicine, San Francisco, California 94143 ABSTRACT. Inactive human renin is found in am- niotic fluid, plasma, and kidney and may be a renin precursor("prorenin"). The mechanism of activation of inactive renin in uiuo is not known. The present study examined the hypothesisthat cathepsinD, a lysosomal pepsinJike endopeptidase may be capable of eliciting activation. Cathepsin D was incubated with inactive renin in human amniotic fluid at pH 4.8 and 22 C for 0-5 h. Marked activation occurred and the reaction displayed first order kinetics with respect to the concen- tration of cathepsin D. The initial velocity of conversion of inactive renin to active renin by cathepsin D was 0.0077o/min/pg cathepsin D. Under identical conditions, the initial velocity of conversion by pepsin was 0.L8Vo/min/pE pepsin. The 25-fold higher potency of pepsin compared with cathepsin D is in accordance with the recognized relative substrate affinities and cat- alytic effrciencies of the two enzymes. Inactive renin in human amniotic fluid seemsto be similar to that found in human kidney and since cathepsin D is present in juxtaglomerular cells, this activation process may have physiological importance. (J Clin Endocrinol Metab 46: 153.1978). FORM OF renin having little or no activ- ity at pH 7.4, but which is converted upon exposure to pH 2.5-4.0to enzymatically active renin has been described in human plasma and amniotic fluid (1-4), as well as in chorion laeve (5), extracts of kidney (3, 6-9), and renin storage granules (10). Morris and Lumbers (11, 12) provided evidencethat acid activation inhuman amniotic fluid isprimarily due to hydrolysis of inactive renin by endog- enouspepsin subsequentto autocatalytic ac- tivation of pepsinogen asthe H* concerqtration increases,with maximal destruction of the peptide inhibitor of pepsin being achieved when the pH reaches 3.5 (13).They proposed that activation may be the result of proteolytic cleavage within a renin precursor.This finding was of biochemical interest as it represented activation of a carboxyl protease by another carboxyl protease. However, it is unlikely that this particular reaction occurs in uiuo. Be- cause an inactive precursor form of renin Received January 28, 1977. Addressreprint requeststo: Dr. B. J. Morris, Depart- ment of Physiology, 5-762, University of California, Schoolof Medicine. San Francisco. California 94143. * This work was supported by the Skaggs Foundation. t The author is a C. J. Martin Research Fellow of the National Health and Medical ResearchCouncil of Aus- tralia. would have to be converted to active enzyme in order to react with renin substrate, Morris and associates (10, 12) proposed that limited hydrolysis of "prorenin" by the intracellular pepsin-like protease, cathepsin D, may acti- vate the precursor before secretion of physio- logically active renin by the juxtaglomerular cells and by cells in the chorion laeve. The aim of the present work was to deter- mine if cathepsin D can activate inactive renin. Human amniotic fluid, which contains a large proportion of inactive renin, a low concentration of renin substrate and low an- giotensinase activity (1, 2) was used. The po- tency of cathepsin D relative to pepsin in the activation was also examined. Materials and Methods Humanamniotic fluid free from contamination by blood was collected afber amniotomy andstored aliquoted at -20 C before use.Cathepsin D (EC 3.4.23.5) from bovine spleenwas purchasedin pow- der form from Sigma Chemjcal Company,St. Louis, Mo. (lot no. 26C-8100); the method of pirification used (14) gave virtually pure enzyme asjudged by electrophoresis. Pepsin (EC 3.4.4.1) fromhog stom- ach mucosawas also purchasedfrom Sigma Chem- ical Company (lot no. 75C-8220) as a twice crystdl- lized and lyophilized powder and was also closeto ro.t