Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate.
We have used native gel electrophoresis followed by fluorogenic peptide overlay to identify multiple forms of rabbit reticulocyte multicatalytic protease (MCP) or 20 S protease, and two forms of rabbit 26 S ubiquitin/ATP-dependent protease. An abundant, fast-migrating 20 S complex (20 SF) possesses modest ability to hydrolyze the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide. In contrast, two minor, slower migrating species cleave the peptide at high rates. A unique 30-kDa polypeptide is associated with one of the active MCPs, and a 160-kDa subunit is associated with the other. Two electrophoretically distinct 26 S proteases can also be isolated from rabbit reticulocyte lysate. The faster migrating form, 26 SF, is more resistant to inactivation by ATP depletion. Despite the differential response to nucleotides and the distinctive electrophoretic mobilities of 26 SF and 26 SS, we have not identified any subunit differences between the two enzymes. In addition to active 26 S proteases, we have discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26 S protease (e.g. molecular masses between 30 and 110 kDa). Incubation of this protein complex with purified MCP and ATP results in the formation of the 26 S proteases.
Hoffman
L
L
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Pratt
G
G
Rechsteiner
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D014452:Ubiquitins / Q000378:metabolism*
D009097:Multienzyme Complexes; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex