title: Changes in the structure and catalytic activities of the bovine pituitary multicatalytic proteinase complex following dialysis.
abstract: The multicatalytic proteinase complex (proteasome) contains at least four distinct active sites catalyzing the degradation of selected chromogenic substrates (trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolyzing activities) and proteins such as beta-casein. Oxidized insulin B chain was recently proposed as a model substrate for protein degradation by the multicatalytic proteinase complex (Dick, L. R., Moomaw, C. R., DeMartino, G. N., and Slaughter, C. A. (1991) Biochemistry 30, 2725-2734). We studied the dialysis-induced activation of the hydrolysis of oxidized insulin B chain by this enzyme. Removal of EDTA from purified preparations of bovine pituitary multicatalytic proteinase complex by dialysis against Tris-HCl buffers led to marked changes in the catalytic properties and structure of the enzyme. Dialysis produced a time-dependent activation of oxidized insulin B chain hydrolysis with predominant cleavage at the Glu13-Ala14 bond. A new chromogenic assay was developed for measurement of this activity. Activation was accompanied by a virtually total inactivation of the chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a loss of the 24-kDa subunit and the appearance of a new band at 21 kDa. Amino-terminal amino acid analysis established that the 21-kDa band was autolytically derived from the 24-kDa subunit. Evidence for partial dissociation and/or aggregation indicated that autolysis destabilizes the complex. By altering the profile of catalytic activities of the multicatalytic proteinase complex, autolysis may serve as a mechanism for regulation of this macromolecule.
authors.affiliation: Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York 10029.
authors.forename: B
authors.initials: B
authors.lastname: Yu
authors.forename: M E
authors.initials: ME
authors.lastname: Pereira
authors.forename: S
authors.initials: S
authors.lastname: Wilk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002863:Chromogenic Compounds / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010902:Pituitary Gland / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex
chemical_list: D002863:Chromogenic Compounds; D007328:Insulin; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex