title: High affinity Zn2+ inhibitory site(s) for the trypsin-like peptidase of the 20S proteasome.
abstract: The effect of Zn(2+) on three major peptidase activities of the 20S proteasome purified from Xenopus oocytes was kinetically investigated. An extremely low concentration of Zn(2+) (microM range) strongly inhibited the trypsin-like activity of the 20S proteasome which was fully recoverable by the addition of EDTA. The concentration of Zn(2+) for half-maximum inhibition (K(0.5)) was 0.60 microM which was at least 10 times lower than that of any other divalent cation tested and essentially the same as for proteasomes purified from various other organisms indicating that the inhibition is highly Zn(2+)-specific, reversible, and common to the proteasome regardless of its source. Zn(2+) at concentrations below 100 microM instantaneously activated the chymotrypsin-like and PGPH activities, and the Zn(2+) concentration for half-maximum activation was found to be 42-48 microM. These activities were time-dependently inactivated by submillimolar concentrations of Zn(2+). The inactivation rates were dependent on the concentration of Zn(2+) and reached a maximum of 1.60-2.40 min(-1) for the three peptidase activities under the conditions used. The Zn(2+) concentration for half-maximum inactivation was found to be 0.70-1.23 mM. This time-dependent inactivation was not reversed by the addition of EDTA or DTT and might not be accompanied by the dissociation of subunits of the 20S proteasome indicating that all activities are inactivated by an identical phenomenon. These results reveal the three types of effects of Zn(2+) on the 20S proteasome.
authors.affiliation: Department of Biology, Faculty of Science, National University Corporation, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Aktar Uzzaman
authors.initials: AU
authors.lastname: Chouduri
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Mishina
authors.forename: Takafumi
authors.initials: T
authors.lastname: Shimizu
authors.forename: Masahito
authors.initials: M
authors.lastname: Yamazaki
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Tokumoto
authors.forename: Shinpei
authors.initials: S
authors.lastname: Yamada
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D061988:Proteasome Inhibitors; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014981:Xenopus; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D061988:Proteasome Inhibitors; D014357:Trypsin; D046988:Proteasome Endopeptidase Complex; D015032:Zinc