title: Isolation and characterization of a novel endogenous inhibitor of the proteasome.
abstract: A novel endogenous inhibitor of the proteasome (high molecular weight multicatalytic protease) has been isolated and characterized from human erythrocytes. After purification by ion-exchange and sizing chromatography, the inhibitor displayed a native molecular mass of approximately 200 kDa and contained a single subunit of 50 kDa with an isoelectric point of 6.9. Although the inhibitor noncompetitively blocks proteolysis of [methyl-14C]-alpha-casein (Ki = 7.1 x 10(-8) M) and inhibits hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC, it did not affect hydrolysis of other peptide substrates, such as MeOSuc-Phe-Leu-Phe-MNA and Z-Ala-Arg-Arg-MNA. To further characterize the 50-kDa inhibitor, a monoclonal antibody (MI-8) was generated that showed specific binding upon Western blot analysis of both native PAGE and SDS-PAGE. Immunoprecipitation with MI-8 specifically removed inhibitor activity against the proteasome. The 50-kDa inhibitor is distinct from a previously described 40-kDa inhibitor of the proteasome (Murakami, K., & Etlinger, J.D. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7588-7592) on the basis of lack of cross-reactivity with MI-8 and dissimilar peptide digest patterns. It is suggested that these endogenous inhibitors may have a role in ATP/ubiquitin-dependent proteolysis and/or other cellular functions involving this protease.
authors.affiliation: Department of Cell Biology and Anatomy, New York Medical College, Valhalla 10595.
authors.forename: X C
authors.initials: XC
authors.lastname: Li
authors.forename: M Z
authors.initials: MZ
authors.lastname: Gu
authors.forename: J D
authors.initials: JD
authors.lastname: Etlinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002384:Catalysis; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D011480:Protease Inhibitors