Chemical modification of the bovine pituitary multicatalytic proteinase complex by N-acetylimidazole. Reversible activation of casein hydrolysis.
The effect of N-acetylimidazole, a mild acetylating reagent, on the catalytic activities and subunit structure of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. The trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide) and the peptidylglutamyl-peptide bond hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) of MPC were rapidly inactivated by N-acetylimidazole, whereas the chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide) was inactivated slowly. However, the hydrolysis of casein was markedly stimulated. Hydrolysis of casein by the acetylated enzyme generated a stable intermediate (21 kDa) which could be further degraded by native MPC. Treatment of acetylated MPC with hydroxylamine reversed the changes in trypsin-like and caseinolytic activities but did not restore the PGP activity. N-Acetylimidazole did not dissociate MPC but altered its migration on nondissociating gels presumably by acetylation of epsilon-amino groups of lysine residues. Hydroxylamine did not alter the gel electrophoretic appearance of the acetylated enzyme. These results indicate that acetylation of thiol or tyrosyl groups changes the trypsin-like and caseinolytic activities, and that amino group acetylation inhibits the PGP activity. Degradation of casein by MPC appears to be a sequential process with initial cleavage catalyzed by a component distinct from the chymotrypsin-like, trypsin-like, and PGP activities. The latter three components likely participate in the secondary proteolysis of the generated intermediates.
Yu
B
B
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York 10029.
Pereira
M E
ME
Wilk
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D010902:Pituitary Gland / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex
D002364:Caseins; D006898:Hydroxylamines; D007093:Imidazoles; D009097:Multienzyme Complexes; D019811:Hydroxylamine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C012160:N-acetylimidazole