3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.
The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.
Pereira
M E
ME
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York 10029.
Nguyen
T
T
Wagner
B J
BJ
Margolis
J W
JW
Yu
B
B
Wilk
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D002850:Chromatography, Gel; D003374:Coumarins / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006868:Hydrolysis; D049934:Isocoumarins; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D010902:Pituitary Gland / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
D002364:Caseins; D003374:Coumarins; D049934:Isocoumarins; D009097:Multienzyme Complexes; D015842:Serine Proteinase Inhibitors; C046235:3,4-dichloroisocoumarin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex