Kinetic studies of the differential effect of detergents on the peptidase activities of the multicatalytic proteinase from rat liver.
We present here a detailed study of the effect of detergents on the three peptidase activities (hydrolysis of the LLVY, ARR, and LLE peptides) of the purified multicatalytic proteinase from rat liver. At Triton X-100 and sodium dodecyl sulfate (SDS) concentrations of 0.1%, all three peptidase activities are inhibited. Lower concentrations of the two detergents (0.01%) do not affect the hydrolysis of the ARR peptide, whereas they behave differently on the hydrolysis of the LLVY and LLE peptides. Triton X-100 inhibits and SDS strongly activates LLVY peptide hydrolysis by decreasing and increasing Vmax, respectively. In the absence of detergents, the saturation curve for the LLE peptide can be analyzed as the result of two components, one showing cooperative (nH = 1.6) with higher affinity (S0.5 = 60 microM) and lower Vmax than a second, noncooperative component (Km = 320 microM). SDS (0.01%) activates LLE peptide hydrolysis by suppressing cooperativity, slightly increasing Vmax, and decreasing the half-saturation concentration (Km = 30 microM) of the enzyme. Triton X-100 (0.01%) also suppresses the cooperativity and decreases the half-saturation concentration (Km = 25 microM) for the LLE peptide; in contrast, it reduces Vmax by inhibition of the low affinity, high Vmax component observed in the absence of detergents. Based on these observations, it can be concluded that both detergents behave like allosteric activators of peptidylglutamyl-peptide hydrolyzing activity and that the multicatalytic proteinase has at least three different classes of active sites: two independent noncooperative sites that catalyze the hydrolysis of trypsin and chymotrypsin-like substrates and one class for peptidylglutamyl-peptide hydrolysis having two components: one cooperative (two or more sites) and one noncooperative.
Arribas
J
J
Departmento de Bioquímica e Instituto de Investigaciones Biomédicas del Consejo Superior de Investigaciones Científicas, Facultad de Medicina de la UAM, Madrid, Spain.
Castaño
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D002793:Cholic Acids / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D005456:Fluorescent Dyes; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D009842:Oligopeptides / Q000138:chemical synthesis; D011092:Polyethylene Glycols / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011919:Rats, Inbred Strains; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology*
D002793:Cholic Acids; D003902:Detergents; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D009842:Oligopeptides; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate