Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins.
The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein.
Mykles
D L
DL
Department of Biology, Colorado State University, Fort Collins 80523.
Haire
M F
MF
D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D000818:Animals; D002364:Caseins / Q000494:pharmacology; D002412:Cations; D010450:Endopeptidases / Q000378:metabolism*; D006358:Hot Temperature; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D009210:Myofibrils / Q000378:metabolism*; D008121:Nephropidae / Q000201:enzymology*; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014336:Troponin / Q000378:metabolism / Q000494:pharmacology
D000597:Amino Acids, Branched-Chain; D002364:Caseins; D002412:Cations; D007976:Leupeptins; D009097:Multienzyme Complexes; D009124:Muscle Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D014336:Troponin; C002101:chymostatin; D010450:Endopeptidases; C032854:leupeptin