Ovine skeletal muscle multicatalytic proteinase complex (proteasome): purification, characterization, and comparison of its effects on myofibrils with mu-calpains.
The latent form of multicatalytic proteinase complex (MCP) was purified to homogeneity from ovine skeletal muscle. The MCP ran as a single band (M(r) 600,000) on nondenaturing polyacrylamide gel (PAGE) and dissociated to a number of subunits (M(r) 21,000 to 31,000) under denaturing and reducing conditions (SDS-PAGE). The proteinase complex was activated reversibly by heating at 60 degrees C and in the presence of SDS. Maximum activation (18-fold) was observed after 2 min at 60 degrees C and there was rapid inactivation beyond 2 min. Maximum proteolytic activity (12.8-fold) occurred in the presence of .25 mM SDS and diminished rapidly at higher SDS concentrations. The MCP was maximally active at pH 7.5 to 8.0 and 45 degrees C using radiolabeled alpha-casein. These and other results (e.g., proteinase inhibitor profiling) indicate that ovine skeletal muscle does indeed contain MCP and that its biochemical properties are the same as MCP isolated from other sources. By using [14C]-casein as a substrate, the specific activities (milligrams of protein degraded/milligrams of proteinase) for mu-, m-calpain, and MCP were 44.0, 59.7, and 2.0, respectively. Purified ovine myofibrils were incubated with mu-calpain or MCP. Classical effects of calpains, which include degradation of Z-disks, titin, desmin, troponin-T, and troponin-I and removal of alpha-actinin, were observed. However, only troponin-C and myosin light chains-2 and -3 were degraded by MCP. Morphologically, MCP had no detectable effect on myofibrils. Results suggest that MCP is not involved in the initial steps of myofibril disassembly. However, its involvement in the degradation of myofilaments remains to be determined.
Koohmaraie
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Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166.
D000818:Animals; D002154:Calpain / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D009210:Myofibrils / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D012756:Sheep / Q000378:metabolism*
D002364:Caseins; D009097:Multienzyme Complexes; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex