Vol. 155, No. 2, 1988 September 15, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 546-553 CALCIUM-ACTIVATED NEUTRAL PROTEASE (CANP), A PUTATIVE PROCESSING ENZYME OF THE NEUROPEPTIDE, KYOTORPHIN, IN THE BRAIN Yoshihiro YOSHIHARA, Hiroshi UEDA, Shinobu IMAJOH*, Hiroshi TAKAGI ~ and Masamichi SATOH Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan *The Institute of Medical Science, The University of Tokyo, Tokyo 180, Japan Received July 26, 1988 Kyotorphin (Tyr-Arg) accumulation in the dialysed synaptosol from the rat brain in the presence of an inhibitor of kyotorphin-degrading enzyme, was maximal at neutral pH. This accumulation was activated by calcium ions, but was inhibited by leupeptin and SH-blocking agents, a finding which suggests the involvement of calcium-activated neutral protease (CANP or ca!pain). In addition, the kyotorphin-precursor protein, being processed by purified ~- or m-CANP, was detected at about 160 kDa on Sephacryl S-300 chromatography of the synaptosol. The present findings seem to be the first evidence for the role of CANP as a processing enzyme of neuropeptide-precursor in nerve terminals. ® 1988 Academic Press, Inc. Kyotorphin (Tyr-Arg), a neurodipeptide isolated from the bovine brain (i), elicits an opioid-like analgesia (2), presumably mediated via a release of met-enkephalin (1,3). Recent studies provided various evidence that kyotorphin may be a neuromodulating peptide, because it is synthesized in nerve terminals (4,5), released by a depolarizing stimuli (6) and **Emeritus Professor of Kyoto University Abbreviations CANP, calcium-activated neutral protease; DTT, dithiothreitol; pCMB, p-chloromercuribenzoate; pAPMSF, (p-amidinophenyl)methane sulfonyl fluoride hydrochloride; E-64, N-[N-(L-3-trans-carboxy- oxiran-2-earbonyl)-L-leucyl]agmatine; EGTA, ethylene glycol bis (~-aminoethylether)-N,N,N',N'-tetraacetic acid. 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 546