The integrity of thiol groups is essential for catalytic efficiency of rat liver cholesterol ester hydrolase either in microsomal membranes or after solubilization.
Neutral cholesterol ester hydrolase from rat liver microsomes was inactivated in a dose and time-dependent manner by classical sulphydryl-reacting reagents such as p-hydroxymercuribenzoic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide, or iodoacetate. The concentrations at which half-maximal inhibition of the native microsomal cholesterol ester hydrolase occurred (IC50) were 15, 68, and 370 mumol/l and 68 mmol/l, respectively. Only partial reactivation of the enzyme was observed under excess dithiothreitol or mercaptoethanol treatment. The stimulation of cholesterol ester hydrolase by the metal ions Ca2+ and Mg2+ was dependent on the integrity of the thiol groups. Solubilization of cholesterol ester hydrolase from membranes preserved its sensitivity towards sulphydryl reagents and thiols, as well as its ability to be activated by Ca2+ and Mg2+. Dithiothreitol, mercaptoethanol, and Ca2+ and Mg2+ provided total protection of the enzyme against inactivation by thiol-reacting reagents. The results indicate that one or more thiol groups are either at the active centre of the native and solubilized forms of rat liver microsomal cholesterol ester hydrolase or are sufficiently near, to interfere with the catalysis when they are reacted.
López de Heredia
M
M
Department of Physiology, Faculty of Medicine, University of the Basque Country, Bilbao, Spain.
Cristóbal
S
S
Hernández
M L
ML
Martínez
M J
MJ
Ochoa
B
B
D000818:Animals; D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000737:chemistry; D005260:Female; D006902:Hydroxymercuribenzoates / Q000737:chemistry; D007461:Iodoacetates / Q000737:chemistry; D019807:Iodoacetic Acid; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008623:Mercaptoethanol / Q000737:chemistry; D008862:Microsomes, Liver / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D002787:Sterol Esterase / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D013997:Time Factors
D002413:Cations, Divalent; D006902:Hydroxymercuribenzoates; D007461:Iodoacetates; D008565:Membrane Proteins; D013439:Sulfhydryl Reagents; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; D002787:Sterol Esterase; D008274:Magnesium; D005033:Ethylmaleimide; D002118:Calcium; D004229:Dithiothreitol; D019807:Iodoacetic Acid