Effect of brief treatment at alkaline pH on the properties of UDP-glucuronosyltransferase.
The kinetic properties of UDP-glucuronosyltransferase were measured after brief treatment of liver microsomes at alkaline pH, followed by assay with p-nitro-phenol as aglycone, at pH 7.5. Enzyme activity increased in a graded fashion as the pH of pretreatment was increased above 8.0, with apparent maximal activation of eight-fold for a pretreatment pH of 11.1. The pH for half maximal activation was 10.6. Brief treatment at alkaline pH prior to assay at pH 7.5 was associated too with a graded conversion of the kinetics of the enzyme from non-Michaelis-Menten to Michaelis-Menten at pH 11.7. Sensitivity to the allosteric modulator, UDP-N-acetylglucosamine decreased as the pH increased. A fifty percent loss of sensitivity to UDP-N-acetylglucosamine-induced activation occurred at pH 10.6. Thus, pretreatment at alkaline pH had irreversible effects on the properties of UDP-glucuronosyltransferase in microsomes. In order to establish the cause for the irreversibility of the changes induced by alkaline pH, microsomes were treated at pH 11.6 prior to purifying UDP-glucuronosyltransferase. Enzyme purified from alkali-treated and untreated microsomes had approximately the same specific activity. More importantly, responses to activation by lipids, and regeneration of allosteric properties were the same for both purified enzymes (from alkali-treated and control microsomes). Pure enzyme was not activated by pretreatment at alkaline pH. We interpret these data to mean that the irreversible effects of alkaline pH on the properties of UDP-glucuronosyltransferase in microsomes were not due to direct effects on the enzyme, but to how the enzyme interacted normally with molecules within the plane of the membrane.
Dannenberg
A
A
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Wong
T
T
Zakim
D
D
D000818:Animals; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D013552:Swine; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000494:pharmacology
D014537:Uridine Diphosphate N-Acetylglucosamine; D014453:Glucuronosyltransferase