Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies.
The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.
Rivett
A J
AJ
Department of Biochemistry, University of Leicester, U.K.
Sweeney
S T
ST
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D007106:Immune Sera; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex