Purification of latent proteasome (20s Proteasome) and demonstration of active proteasome in goldfish (Carassius aurmus) oocyte cytosol

Toshinobu Tokumoto, Michiyasu Yoshikuni, Hiroko Kajiura, Yoshitaka Nagahama, Masakane Yamashita

Research output: Contribution to journalArticle

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Abstract

The cytosol fraction (150,000 g supernatant) of goldfish ovary hydrolyzed a fluorogenic peptide, succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, a well-known substrate for proteasome, regardless of the addition of SDS to the reaction mixture. Four steps of column chromatography resulted in a 135-fold purification of proteasome from this supernatant. Both SDS-independent and SD S-dependent hydrolyzing activities co-migrated during purification. However, the SDS-independent activity was markedly reduced during purification. The purified proteasome possessed faint hydrolyzing activity (chymotrypsin-, trypsin-, and V83 protease-like activity) even in the absence of SDS; the chymotrypsin- and V8 proteaselike activities were significantly increased in the presence of SDS. Its molecular weight and sedimentation coefficient were estimated to be 620 kDa and 19.2 S, respectively. Three kinds of monoclonal antibodies were raised against the purified proteasome. Western blot analyses revealed that these antibodies recognized a single species of protein on native PAGE, but recognized several subunits ranging in molecular mass from 23.5 to 31.5 kDa on SDS-PAGE. Cytosol fractions containing the SDS-independent activity had a band which migrated slower than that ofpurified proteasome. The SDS-independent protease activity was depleted when the cytosol fraction was immunoprecipitated with the anti-proteasome antibody. From these structural and enzymatical properties, it is concluded that the purified proteasome corresponds to latent proteasomes (20 S proteasomes) reported in other eukaryotic cells. Further, the results demonstrate that goldfish oocytes contain an active form of proteasome which can hydrolyze its substrates in the absence of activators.

Original languageEnglish
Pages (from-to)173-186
Number of pages14
JournalBiomedical Research (Japan)
Volume16
Issue number3
DOIs
Publication statusPublished - Jan 1 1995

Fingerprint

Goldfish
Proteasome Endopeptidase Complex
Cytosol
Oocytes
Purification
Demonstrations
Peptide Hydrolases
Native Polyacrylamide Gel Electrophoresis
Column chromatography
Antibodies
Chymotrypsin
Molecular mass
Eukaryotic Cells
Substrates
Sedimentation
Chromatography
Polyacrylamide Gel Electrophoresis
Anti-Idiotypic Antibodies
Ovary
Molecular Weight

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Purification of latent proteasome (20s Proteasome) and demonstration of active proteasome in goldfish (Carassius aurmus) oocyte cytosol. / Tokumoto, Toshinobu; Yoshikuni, Michiyasu; Kajiura, Hiroko; Nagahama, Yoshitaka; Yamashita, Masakane.

In: Biomedical Research (Japan), Vol. 16, No. 3, 01.01.1995, p. 173-186.

Research output: Contribution to journalArticle

Tokumoto, Toshinobu ; Yoshikuni, Michiyasu ; Kajiura, Hiroko ; Nagahama, Yoshitaka ; Yamashita, Masakane. / Purification of latent proteasome (20s Proteasome) and demonstration of active proteasome in goldfish (Carassius aurmus) oocyte cytosol. In: Biomedical Research (Japan). 1995 ; Vol. 16, No. 3. pp. 173-186.
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