Dissociation and reassociation of the bovine pituitary multicatalytic proteinase complex.
The eukaryotic multicatalytic proteinase complex (proteasome) is a high molecular mass enzyme which contains 13-15 nonidentical subunits of similar size (molecular masses of 21-31 kDa), but differing widely in net charge (isoelectric points ranging from 3 to 10). At least four catalytic components termed chymotrypsin-like, trypsin-like, peptidylglutamyl peptide-hydrolyzing, and caseinolytic are associated with the proteinase. The catalytic nature of the components is unknown, since sequences of cloned subunits bear no homology to known proteinases and proteolytically active subunits have not been isolated. Analysis of the relationship between structure and catalytic function would be greatly facilitated if a means for reversibly dissociating and reassociating the proteinase were available. We provide the first evidence of reassembly of dissociated multicatalytic proteinase complex into a functional molecule. Incubation with the organic mercurial, p-chloromercuribenzoic acid disrupts in a concentration-dependent manner the quaternary structure of the enzyme, leading to formation of a heterogeneous population of subunits. Dissociation of the complex coincides with progressive loss of chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. The caseinolytic activity of the residual undissociated enzyme is markedly activated. Exposure of the dissociated enzyme to dithiothreitol restores the catalytic profile and reassociates the enzyme. Evidence for catalytically active subcomplexes was not obtained indicating that structural integrity may be necessary for expression of all defined activities.
Figueiredo-Pereira
M E
ME
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York 10029.
Yu
B
B
Wilk
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism*; D010902:Pituitary Gland / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D020245:p-Chloromercuribenzoic Acid; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D004229:Dithiothreitol