Purification of an 11 S regulator of the multicatalytic protease.
We have identified and purified a protein complex from human red blood cells that activates the multicatalytic protease (MCP). The complex, which we call the regulator, sediments at 11 S and is composed of 30-kDa subunits. The regulator does not hydrolyze fluorogenic peptides, but when multicatalytic protease and regulator are combined, MCP cleaves succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and Leu-Leu-Glu-p-nitroanilide as much as 60-fold faster. Hydrolysis of several other fluorogenic peptides is stimulated to a lesser extent, and activated MCP does not degrade ubiquitin-lysozyme conjugates, bovine serum albumin, or lysozyme. Latent and activated forms of MCP display similar sensitivity to protease inhibitors, suggesting that activation does not generate new kinds of catalytic sites. In addition, ATP suppresses peptide hydrolysis by activated and latent MCPs to the same extent. Activation involves binding of regulator to MCP, and activated MCP migrates slower on native acrylamide gels. Dissociation of the MCP regulator complex during prolonged sedimentation on glycerol gradients releases active regulator and MCP molecules capable of being reactivated. Moreover, two-dimensional electrophoresis does not reveal changes in MCP or regulator subunits following activation. Thus, activation appears to result from reversible association of regulator subunits with MCP.
Dubiel
W
W
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Pratt
G
G
Ferrell
K
K
Rechsteiner
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D014461:Ultracentrifugation
D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex