Activation of the 20S Proteasome of Xenopus Oocytes by SDS : Evidence for the Substrate-Induced Conformational Change Characteristic of Trypsin-Like Peptidase

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概要

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• The 20S proteasome of eukaryotic cells has at least three distinct peptidase activities(trypsin-like, chymotrypsin-like and peptidylglutamylpeptide(PGP)hydrolase activities). These peptidases are latent and require appropriate activators. SDS has been widely used as an activator of these peptidases, but the mechanism of its activation remains unresolved. In this study, we investigated the kinetics of the SDS-activated hydrolysis of the above three types of peptidase of the 20S proteasome purified from Xenopus oocytes. When the reaction was started by simultaneous adding both SDS and substrtate, maximal rates of hydrolysis were reached after appreciable lag phases with the trypsin-type substrate [t-butyloxycarbonyl-Leu-Arg-Arg-4-methylcoumaryl-7-amide(Boc-LRR-MCA)], but no such lag phases were observed with the chymotrypsin-type and PGP hydrolase-type substrates [succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide(Suc-LLVY-MCA), and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide(Cbz-LLE-2NA), respectively]. Similarly, changes in the hydrolysis rate to a reduced level upon dilution of SDS occurred after an appreciable lag phase again in the trypsin-like peptidase, but not in the other types. The lag phase characteristic of the trypsin-like peptidase was dependent on the substrate concentration. Thus, the lag phase was less discernible at very low concentrations of the substrate(e.g.at concentrations in the order of 1/100 of the Km value), but became more conspicuous with the increases in the substrate concentration. This lag phase also vanished upon preincubation of the activator(SDS)for a short period of 5 sec. These results suggest that the formation of the exzyme-substrate complex in the trypsin-like reaction induces a conformational chagte in the enzyme which makes the SDS activator site(s)in an occluded form, reducing the rates of SDS binding and dissociation.

• 社団法人日本動物学会の論文

著者

• Yamada Junko

  Department of Neurophysiology, Hirosaki University Graduate School of Medicine

• Sato Kenji

  Department Of Food Sciences And Nutritional Health Kyoto Prefectural University

• Sato Katsuaki

  Central Research Laboratories Of Ajinomoto Co. Inc.

• Yamada Junko

  Department Of Biology And Geosciences Faculty Of Science Shizuoka University

• Kubota Satoshi

  Division Of Human Health And Medical Science Graduate School Of Kuroshio Science Kochi University

• ISHIKAWA Katsutoshi

  Department of Biology, Faculty of Science, Shizuoka University

• TOKUMOTO Toshinobu

  Department of Biology, Faculty of Science, Shizuoka University

• YAMADA Shigehiro

  ORYNOVA K. K.

• Sato K

  Department Of Food Sciences And Nutritional Health Kyoto Prefectural University

• Kubota S

  Division Of Human Health And Medical Science Graduate School Of Kuroshio Science Kochi University

• Yamada S

  Department Of Biological Chemistry College Of Bioresource Sciences Nihon University:(present Address

• Sato Kentaro

  Department Of Otorhinolaryngology Kanagawa Dental College

• Yamada Shinpei

  Department Of Biorogy Faculty Of Science Shizuoka University

• Yamada Shinpei

  Department Of Biology And Geoscience Faculty Of Science Shizuoka University

• Ishikawa K

  Department Of Biology And Geosciences Faculty Of Science National University Corporation Shizuoka Un

• Yasutomi Masumi

  Laboratory of Biology, Aichi Medical University

• Yasutomi Masumi

  Lab, of Biology, Aichigakuin university

• Yasutomi Masumi

  Lab Of Biology Aichigakuin University

• Yasutomi Masumi

  Laboratory Of Biology Aichi Medical University

• Yamada Junko

  Department Of Biotechnology Graduate School Of Engineering Nagoya University

• Yamada S

  Department Of Biology And Geosciences Faculty Of Science Shizuoka University

• Ishikawa Katsutoshi

  Department Of Biology And Geosciences Faculty Of Science Shizuoka University

• Tokumoto Toshinobu

  Department Of Biology And Geosciences Faculty Of Science National University Corporation Shizuoka Un

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