Kinetic Studies of the Branched Chain Amino Acid Preferring Peptidase Activity of the 20S Proteasome:  Development of a Continuous Assay and Inhibition by Tripeptide Aldehydes andclasto-Lactacystin β-Lactone

Biochemistry ◽  
1998 ◽  
Vol 37 (21) ◽  
pp. 7792-7800 ◽  
Author(s):  
Teresa A. McCormack ◽  
Amy A. Cruikshank ◽  
Louis Grenier ◽  
Francesco D. Melandri ◽  
Sandra L. Nunes ◽  
...  
Keyword(s):  
Amino Acid ◽  
Kinetic Studies ◽  
20S Proteasome ◽  
Peptidase Activity ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Continuous Assay

scholarly journals Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins

1995 ◽  
Vol 306 (1) ◽  
pp. 285-291 ◽  
Author(s):  
D L Mykles ◽  
M F Haire
Keyword(s):  
Amino Acid ◽  
Myofibrillar Proteins ◽  
Significant Finding ◽  
Peptidase Activity ◽  
Multicatalytic Proteinase ◽  
Branched Chain Amino Acid ◽  
Endogenous Protein ◽  
Proteolytic Activities ◽  
Branched Chain

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.

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