Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase. Evidence for thiol-dependent allosteric modulation of enzyme activity.

摘要: Rat liver microsomes devoid of free thiols were prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer the presence 30 microM leupeptin. The activation 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by GSH and dithiothreitol (DTT) these was studied compared to enzyme that solubilized freeze-thawing. An increase Vmax observed, for two preparations, with increasing concentrations activating thiols. Reactions GSH-activated microsomal NADPH show sigmoidal kinetics a Hill coefficient 2.01 +/- 0.07 at 2-4 mM GSH. Increase resulted gradual change towards Michaelis-Menten kinetics, 1.08 0.03 calculated 25 Activation DTT yielded similar results except 1.1 observed already 2.5 DTT. Normal HMG-CoA all concentrations. Solubilization widely used freeze-thaw procedure abolished cooperative pattern, normal 1.0 regardless concentration. These are compatible model which activity is GSH-dependent, allosterically modulated under physiological hepatic conditions. In addition, assay conditions, using high or employment highly purified soluble precluded observation suggested model.