Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization.
A ubiquitin (Ub)/ATP-dependent proteolytic complex (26S proteasome) purified from rabbit skeletal muscle was dissociated into two subcomplexes, a 20S proteasome and a regulatory subunit complex, by preparative non-denaturing polyacrylamide gel electrophoresis (PAGE). The isolated regulatory subunit complex preparation gave a single broad band on analytical non-denaturing PAGE, and several bands ranging between 33 and 110 kDa on SDS-PAGE. This complex was found to consist of about 20 subunits on the basis of two-dimensional PAGE, the pattern of which appeared identical or very similar to that of the 33-110 kDa 26S proteasome subunits. The apparent molecular mass of the complex was estimated to be 1100 kDa by Ferguson plot analysis and also by Superose 6 gel filtration. Unlike the 26S proteasome, neither ATPase activity nor protease activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Phe-Ser-Arg-MCA, Z-Leu-Leu-Glu-beta NA, [14C]-casein, [125I]-lysozyme and Ub-[125I]-lysozyme were significantly detectable in the regulatory subunit complex. This complex was found to be capable of associating with itself in MgATP-dependent manner. These results suggest that a regulatory subunit complex dissociated from the 26S proteasome comprises all the higher molecular mass subunits of the 26S proteasome, and has no detectable ATPase and protease activities, although the homo-oligomerization occurs in an ATP-dependent fashion.
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
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Akaishi
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Yokosawa
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Sawada
D000595:Amino Acid Sequence; D000818:Animals; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000032:analysis*; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism
D009097:Multienzyme Complexes; D014452:Ubiquitins; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease