A NOVEL CHYMOTRYPSIN-LIKE COMPONENT OF THE MULTICATALYTIC PROTEINASE COMPLEX OPTIMALLY ACTIVE AT ACIDIC PH

被引:37
作者
FIGUEIREDOPEREIRA, ME [1 ]
CHEN, WE [1 ]
YUAN, HM [1 ]
WILK, S [1 ]
机构
[1] CUNY MT SINAI SCH MED,DEPT PHARMACOL,NEW YORK,NY 10029
关键词
PROTEOLYTIC ENZYME; ACIDOSIS; INHIBITOR;
D O I
10.1006/abbi.1995.1137
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The multicatalytic proteinase complex (MPC) or proteasome is a multimeric, high-molecular-weight (700,000), extralysosomal proteolytic enzyme found in eukaryotes and in archaebacteria. Its multiple catalytic sites grant it a broad cleavage specificity toward short peptides and protein substrates. The pH optima of the catalytic activities of MPC are in the neutral or slightly alkaline range. We present here evidence for cryptic catalytic components of MPC optimally active at an acidic pH. Studies with a hydrophobic fluorescent probe provide direct evidence for conformational changes brought about by exposing the complex to an acidic environment. One of the newly described components, designated ''acidic chymotrypsin-like activity,'' cleaves the Leu-2-naphthylamide bond in the substrate Boc-Val-Glu-Ala-Leu-2-naphthylamide. Compared with the classical ''neutral'' chymotrypsin-like activity defined by cleavage of the Leu-p-nitroanilide bond in Z-Gly-Gly-Leu-p-nitroanilide, the newly described component is not inhibited by monovalent cations and is less sensitive to the peptidyl aldehyde Z-Gly-Gly-leucinal, an inhibitor of the neutral chymotrypsin-like activity. In addition, we describe the properties of a novel potent peptidyl aldehyde, Z-Ile-GIu(OtBu)-Ala-leucinal, which is an inhibitor of both the acidic and neutral chymotrypsin-like activities of MPC, with IC50 values of 0.25 and 6.5 mu M, respectively. In the presence of 65 mu M of the newly synthesized peptidyl aldehyde, other MPC components such as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities were decreased only by 14 and 9%, respectively. The hydrophobicity, potency, and specificity of Z-Ile-GIu(OtBu)-Ala-leucinal toward the chymotrypsin-like activities of the complex make it a valuable pharmacological tool with which to investigate the physiological roles of MPC. (C) 1995 Academic Press, Inc.
引用
收藏
页码:69 / 78
页数:10
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