Biochcmicnl Phnr~~~~bgy, Vol. 36. NO. 9. pp. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC 1435-1446, 1987 Primed in Great Britain. ooo6-29q87 s3.00 + 0.00 Pqamon hmmls Ltd. HUMAN LIVER PHENOL SULFOTRANSFERASE: ASSAY CONDITIONS, BIOCHEMICAL PROPERTIES AND PARTIAL PURIFICATION OF ISOZYMES OF THE THERMOSTABLE FORM* NORMAN R. C. CAMPBELL,t JON A. VAN LOON and RICHARD M. WEINSHILBOUMS Clinical Pharmacology Unit, Department of Pharmacology, Mayo CIinic/Mayo Foundation, Rochester, MN 55905, U.S.A. (Received 21 October 1985; accepted 26 September 1986) Abstract-Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and ncurotransmittcrs. Human platelets and brain contain at least two forms of PST. One form is relatively thermolabile (TL) and catalyzes the sulfate conjugation of monoamines such as dopamine. The other is thermostable (TS) and catalyzes the sulfation of “simple” phenols such as phenol and p- nitrophenol. We found that homogenates of human liver also contain two forms of PST that are similar to brain and platelet TI. and TS PST with regard to substrate specificities, thermal stabilities and sensitivities to inhibitors. Optimal conditions were determined for the assay of these two activities in human liver homogenates. The apparent K, of liver homogenate TL PST for dopamine was 27 @I. The apparent K,,, of the TS form of the eruyme for p-nitrophenol was 0.94 $4. Human liver TS PST also catalyzed the sulfate conjugation of dopamine, but with an apparent K,,, of 5 mM, over two orders of magnitude higher than that of TL PST. Two different peaks of TS PST activity were separated from the TL activity by ion exchange chromatography of human liver preparations. Both peaks of TS PST activity were partially purified and characterized. Both had similar substrate specificities and inhibitor sensitivities. K,,, values of TS PST peak I for p-nitrophenol and for 3’-phosphoadenosineJ’-phos- phosulfate were 0.91 and 0.86 pM, respectively, while the K,,,values of TS PST peak II for these two cosubstrates for the reaction were 0.43 and 0.64 pM, respectively. However, the TS PST activity in peak II was significantly more thermolabile than was the activity in peak I. These results are compatible with the conclusion that human liver homogenates contain at least two forms of PST, fotms with properties similar to those of TS and TL PST in homogenates of human cerebral cortex and platelets. In addition, human liver contains two isoxymes of TS PST. Sulfate conjugation catalyzed by phenol sulfotransferase (EC 2.8.2.1, PST) is an important metabolic pathway for many phenolic and catechol drugs and neurotransmitters [l-3]. Human platelets and brain contain at least two forms of PST, forms that differ in their substrate snecificities. thermal stabilities, inhibitor sensitivities and regulation 14-101. One form is relativelv thermolabile (TL) . 2 2 \ I and catalyzes the sulfate conjugation of micromolar concentrations of dopamine and other catechol and phenol monoamines. The other form is thermostable (TS) and catalyzes the sulfate conjugation of micro- molar concentrations of phenol, p-nitrophenol and other “simple” phenols [6,7]. However, at higher concentrations phenol and p-nitrophenol can also serve as substrates for TL PST (6,7]. The TS form l This work was suooorted in Dart bv NIH Grants GM 28157 and GM 357% and by NI’H Contract ES 55110. 1 Dr. Campbell is a Merck Sharpe & Dohmc Intema- tional Fellow in Clinical Pharmacolonv. Present address: Faculty of Medicine, Health Science~Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada AlB 3V6. $ Dr. Weinshilboum is a Burroughs Wellcome Scholar in Clinical Pharmacology. Reprint requests should be addressed to this author. is more sensitive to inhibition by 2,6-dichloro-4- nitrophenol (DCNP) than is TL PST [5,6,8]. There are also individual familial variations in’the thermal stability of TS PST in the human platelet [ll], vari- ations that are correlated with individual differences in the thermal stability of TS PST in human cerebral cortex [12]. Human platelet PST has been studied extensively because of the possibility that its bio- chemical properties and regulation might reflect those of the enzyme in less accessible drug and neurotransmitter metabolizing tissues [3]. This hypothesis has already been shown to be valid in the case of TS PST activity in the human platelet and cerebral cortex [12]. The liver plays an important role in the metabolism of both exogenous and endogenous phenolic substances. Our experiments were performed to determine whether the human liver contains PST activities with properties similar to those of the enzyme in the human platelet and brain. MATERLUS AND METHODS Liver tissue acquisition and preparation Liver tissue was obtained from eighteen male and eight female patients undergoing clinically indicated partial hepatectomies for the removal of either pri- 143.5 BP 3e:9-0