Use of serine-protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex.
The multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy-group side of basic, hydrophobic or acidic amino acid residues. Originally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for so-called trypsin-like activity with Boc-Leu-Ser-Thr-Arg-NH-Mec (Mec, 4-methylcoumarin; Boc, t-butoxycarbonyl), for chymotrypsin-like activity with Ala-Ala-Phe-NH-Mec and Suc-Leu-Leu-Val-Tyr-NH-MEc (Suc, succinyl), and peptidyl-glutamylpeptide hydrolase activity with Cbz-Leu-Leu-Glu-Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin-like activity and two for each of the others. The activities were tested with a variety of serine-protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4-(2-amino-ethyl)benzenesulphonyl fluoride, which selectivity inactivates the trypsin-like activity, and 3,4-dichloroisocoumarin which inhibits chymotrypsin-like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133-4141] of peptidylglutamylpeptide hydrolase activity. The three activities inhibited by 3,4-dichloroisocoumarin can easily be distinguished by the effects of chymostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.
Djaballah
H
H
Department of Biochemistry, University of Leicester, England.
Harness
J A
JA
Savory
P J
PJ
Rivett
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D003374:Coumarins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D049934:Isocoumarins; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017208:Rats, Wistar; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D003374:Coumarins; D049934:Isocoumarins; D009097:Multienzyme Complexes; D015842:Serine Proteinase Inhibitors; D007531:Isoflurophate; C046235:3,4-dichloroisocoumarin; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex