Bovine Spleen Multicatalytic Proteinase Complex (Proteasome)
REPLACEMENT OF X, Y, AND Z SUBUNITS BY LMP7, LMP2, AND MECL1 AND CHANGES IN PROPERTIES AND SPECIFICITY*
- From the Departments of ‖Pharmacology,§Biochemistry, and ¶Medicine, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 and ‡University of Camerino, 62032 Camerino, Italy
Abstract
Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-γ-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased K m, a highly increased catalytic efficiency (k cat), and a 80–180 times greater specificity constant (k cat/K m) toward substrates with either branched chain or aromatic amino acid residues in the P1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P3position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-γ-inducible subunits.
Footnotes
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↵* This work was supported by National Institutes of Health Grants DK25377 (to M. O.) and KO8HL02835 (to C. C.) and by a postdoctoral fellowship from Signal Pharmaceuticals Inc. (to A. M. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵** To whom correspondence should be addressed: Box 1215, Dept. of Pharmacology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029. Tel.: 212-876-5211; Fax: 212-831-0114.
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↵1 MPC, multicatalytic proteinase complex; BrAAP, branched chain amino acid preferring; ChT-L, chymotrypsin-like; DCI, 3,4-dichloroisocoumarin; HPLC, high pressure liquid chromatography; IFN, interferon; MCA, 7-amino-4-methylcoumaryl amide; 2NA, 2-naphthylamide; pNA, p-nitroaniline;pAB, p-aminobenzoate; PGPH, peptidylglutamyl-peptide hydrolyzing; PAGE, polyacrylamide gel electrophoresis; SNAAP, small neutral amino acid preferring; T-L, trypsin-like; Z or Cbz, benzyloxycarbonyl. Conventional one- or three-letter abbreviations are used for amino acids.
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↵2 The nomenclature of Schechter and Berger (50) is used to describe the position (P) of the amino acid residues of the substrate in relation to the scissile bond.
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- Received November 14, 1996.
- Revision received February 21, 1997.
