Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization.

T Akaishi; H Yokosawa; H Sawada

doi:10.1016/0304-4165(95)00122-0

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Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization.

T Akaishi·H Yokosawa·H Sawada

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Abstract

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.

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Amino Acid Sequence Animals Cysteine Endopeptidases Electrophoresis, Gel, Two-Dimensional Enzyme Activation Molecular Sequence Data Multienzyme Complexes Muscle, Skeletal Peptide Hydrolases Proteasome Endopeptidase Complex Rabbits Substrate Specificity Ubiquitins Journal Article Research Support, Non-U.S. Gov't

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T Akaishi, H Yokosawa, H Sawada. 1995-12-14. Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization.. https://doi.org/10.1016/0304-4165(95)00122-0

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