Regulation of the peptidylglutamyl-peptide hydrolyzing activity of the pituitary multicatalytic proteinase complex.
The finding that the activity of the multicatalytic proteinase complex (MPC) is greatly activated by low concentrations of sodium dodecyl sulfate (SDS) and fatty acids led to the proposal that the proteolytic activity of the complex is latent and that activation is needed for expression of full activity. Kinetic examination of the nature of the latency with Cbz-Leu-Leu-Glu-2-naphthylamide, a substrate cleaved by the peptidylglutamyl-peptide hydrolyzing activity (PGPH activity) of the complex, showed that plots of velocity versus substrate concentration yield sigmoidal curves, implying the presence of two or more substrate binding sites and the presence of cooperative interactions between the sites. Hill plots of log [v/(Vmax-v)] versus log [S] gave slopes with a Hill coefficient of 2.2-2.4, suggesting that more than two subunits are expressing the PGPH activity. At saturating substrate concentrations, SDS and lauric acid exposed a masked component of PGPH activity that was about equal in magnitude to the overt activity measured in the absence of these detergents, showing that under the latter conditions only about half of the enzyme activity is expressed. Activation by SDS and lauric acid was greater at low than at high substrate concentrations and was associated with a shift of the substrate concentration at half-Vmax (apparent Km) toward lower values. The decrease in the apparent Km in the presence of SDS (but not in the presence of lauric acid) was associated with a decrease in cooperativity. The presence of at least two distinct PGPH activity components with different reactivities was also indicated by the finding of two distinct inactivation rate constants in reactions with 3,4-dichloroisocoumarin, an irreversible inhibitor of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Orlowski
M
M
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York.
Cardozo
C
C
Hidalgo
M C
MC
Michaud
C
C
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005971:Glutamates; D006868:Hydrolysis; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D010902:Pituitary Gland / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity
D005971:Glutamates; D009097:Multienzyme Complexes; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex