An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts.
Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.
McGuire
M J
MJ
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
Reckelhoff
J F
JF
Croall
D E
DE
DeMartino
G N
GN
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D009113:Muramidase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D002364:Caseins; D009097:Multienzyme Complexes; D011506:Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D009113:Muramidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex