Vol. 294, Issue 1, 80-88, July 2000

Phenacetin Deacetylase Activity in Human Liver Microsomes: Distribution, Kinetics, and Chemical Inhibition and Stimulation

Shoji Kudo, Ken Umehara, Masakiyo Hosokawa, Gohachiro Miyamoto, Kan Chiba and Tetsuo Satoh

Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan (S.K., K.U., G.M.); Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan (M.H., K.C.); and Biomedical Research Institute, HAB Discussion Group, Chiba, Japan (T.S.)

Microsomal and cytosolic phenacetin deacetylase activities were examined in human liver and kidneys. Kinetic properties of the activities were also studied in human liver microsomes. Phenacetin deacetylase activity was predominantly localized in the liver microsomal fraction. The specific activities of phenacetin deacetylation in liver cytosol and in kidney microsomes and cytosol were all less than 5% of that in liver microsomes. In human liver microsomes, Eadie-Hofstee plots for phenacetin deacetylation were monophasic, indicating a single-enzyme catalytic reaction. The Michaelis-Menten parameters, K_m and V_max, for the deacetylation were 4.7 mM and 5.54 nmol/min/mg of protein, respectively. The intrinsic clearance, calculated as V_max/K_m, was 1.18 µl/min/mg of protein. Although the organophosphate bis(4-nitrophenyl)phosphoric acid markedly inhibited the reaction in human liver microsomes, the activity has a tolerance to the treatment of phenylmethylsulfonyl fluoride, a serine hydrolase inhibitor. Prazosin, a peripheral ₁-adrenergic antagonist, noncompetitively inhibited the phenacetin deacetylation with a K_i value of 19.0 µM. Flutamide, a nonsteroidal androgen receptor antagonist, stimulated the activity by up to 349%. This increase was accompanied by a decrease in the K_m value and no change in the V_max value, resulting in an increase in the intrinsic clearance by up to 700% of the control. These results suggest that the phenacetin deacetylase localized in human liver microsomes has not only a catalytic site but also a negative and/or positive modulation site or sites.