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|dc.description.abstract||The multicatalytic proteinase complex (MCP) or proteasome is a high molecular mass (650 kDa) proteinase found in all eukaryotes and a similar particle has been purified from the archaebacteria Thermoplasma acidophilum. MCP can cleave peptide bonds on the carboxyl side of basic, hydrophobic, or acidic amino acid residues, and these activities have been referred to as "trypsin-like", "chymotrypsin- like", and "peptidylglutamyl-peptide hydrolase (Glu-X)" activities, respectively. In this study, the Glu-X activity, assayed with the substrate Z-Leu- Leu-Glu-naphthylamide was investigated. Kinetic studies have shown that it is composed of two distinct components: (a) a noncooperative component (LLE1) obeying Michaelis-Menten kinetics, and (b) a cooperative component (LLE2) exhibiting sigmoidal behaviour. The LLE2 component can easily be distinguished from that of the LLE1 component by the effect of inhibitors, divalent metal ions, KC1, and heat treatment. The addition of 1 mM MnC12 stimulates both components and permits saturation of MCP with substrate at concentrations below the solubility limits of the substrate, under these conditions, a Hill coefficient of 5.2 was calculated for the LLE2 component. Using serine protease inhibitors, peptides as model substrates, and other effectors as probes for the different activities, it was found that (a) the proteinase is in fact a novel type of serine protease, (b) there seems to be at least seven distinct proteolytic activities associated with the complex, and (c) the specificity of the various activities is more complex than first proposed, where the "trypsin-like" activity can cleave after tyrosine residues. Possibility of conformational changes associated with the activation by MnCl2, and with the action of different effectors of the Glu-X were investigated by sedimentation velocity analysis, dynamic light scattering measurements, and electron microscopy of negatively stained preparations. The results provide a direct evidence for conformational changes associated with the activation by MnCl2. Electron microscopic observations indicate a transition from a compact to an extended conformation of the complex in the presence of MnCl2. The hydrodynamic results also provide a structural evidence for the observed positive cooperativity exhibited by the LLE2 component. A correlation between activity and shape of the conformational state of MCP was obtained.||en|
|dc.rights||Copyright © the author. All rights reserved.||en|
|dc.title||Peptidylglutamyl-peptide hydrolase activity of the multicalytic proteinase complex.||en|
|dc.publisher.institution||University of Leicester||en|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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