Isolation and characterisation of two chymotrypsins from the midgut of Locusta migratoria

Two chymotrypsin isozymes (CTR 1 and CTR 2) from the midgut lumen of Locust a migratoria have been identified and purified. MALDI-TOF mass spectrometry revealed an M-r of 22 679 (+/-30) for CTR 1 and 22 592 (+/-30) for CTR 2. Both chymotrypsins hydrolysed S-(Ala)(2)ProPhe-pNA (CTR 1: K-m=0.29+/-0.01 mM, V-max=83.0+/-1.4 U/mg; CTR 2: K-m=0.42+/-0.01 mM, V-max=48.9+/-1.1 U/mg ) and S-(Ala)(2)ProLeu-pNA (CTR 1: K-m=0.50+/-0.04 mM, V-max 1.7+/-0.1 U/mg ; CTR 2: K-m=1.12+/-0.08 mM, V(max)11.4+/-0.6 U/mg), but neither enzyme hyd rolysed BTpNA, S-Phe-pNA, Ac-Leu-pNA or S-(Ala)(3)-pNA. CTR 1 and CTR 2 act ivities were effectively inhibited by AEBSF, PMSF, TPCK, chymostatin, SBTI and BPTI. Using S-(Ala)(2)ProPhe-pNA as the substrate, CTR 1 gave optimal a ctivity between pH 8.0 and 10.0, while CTR 2 was optimally active over the range pH 8.0-11.0. The N-terminal 15 amino acids of the purified chymotryps ins were determined, revealing their unique sequences which are also differ ent from another, previously characterised Locusta chymotrypsin. (C) 1999 E lsevier Science Ltd. All rights reserved.