Biochemical studies on the bovine pituitary multicatalytic proteinase complex (MPC): Evidence for a new distinct catalytic component and mechanism of dialysis activation.
Item
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Title
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Biochemical studies on the bovine pituitary multicatalytic proteinase complex (MPC): Evidence for a new distinct catalytic component and mechanism of dialysis activation.
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Identifier
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AAI9325169
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identifier
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9325169
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Creator
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Yu, Bo.
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Contributor
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Adviser: Sherwin Wilk
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Date
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1993
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Neuroscience | Health Sciences, Pharmacology
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Abstract
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The multicatalytic proteinase complex (MPC) is a 700 KDa proteinase containing multiple polypeptide subunits (Mr = 24,000-31,000) arranged as four rings in a cylinder-shaped particle. MPC contains at least three distinct catalytic activities, named as chymotrypsinlike, trypsinlike and peptidylglutamyl peptide bond (PGP) hydrolysing. In addition, MPC hydrolyzes selected proteins such as {dollar}\alpha{dollar} and {dollar}\beta{dollar} crystallin, oxidized glutamine synthetase, oxidized hemoglobin, and casein. This latter activity is referred to in this thesis as caseinolytic activity. A characteristic feature of the caseinolytic activity is its latency which is thought to be an important mechanism for protection of the cell from uncontrolled proteolysis. Activation has been achieved by treatment with SDS, polycations, dialysis against distilled water and by heating.;The relationship of the activities of MPC toward synthetic substrates to the caseinolytic activity has not been clarified. The mechanism for the dialysis activation remains unclear. The studies described in this thesis provide strong evidence that the caseinolytic activity is distinct and elucidate the mechanism of the dialysis activation.;After treatment with N-acetylimidazole (a mild acetylating reagent), the trypsinlike and PGP activities of MPC are markedly inhibited, the chymotrypsinlike activity is almost unaffected, but the caseinolytic activity is increased dramatically. This suggests that the active site responsible for degradation of {dollar}\beta{dollar}-casein is distinct from the other three. Furthermore, hydrolysis of {dollar}\beta{dollar}-casein by the acetylated enzyme generated a stable intermediate which could be further degraded by native MPC. Thus, the degradation of casein by MPC seems to be a sequential process with initial cleavage catalyzed by a distinct caseinolytic component. Removal of EDTA from MPC by dialysis against Tris-HCl buffers produced a time dependent activation of oxidized insulin B chain hydrolysis with predominant cleavage at the Glu{dollar}\sp{13}{dollar}-Ala{dollar}\sp{14}{dollar} bond. After dialysis, MPC loses its chymotrypsinlike, trypsinlike, and PGP activities almost completely, and at least one subunit is autolyzed. The resulting molecule is partially dissociated indicating 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. Other unpublished studies support the independence of the caseinolytic component and point to the existence of yet another distinct activity which can initiate the degradation of the oxidized B chain of insulin.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
