Alkane thiol S-methyltransferase: An enzymatic process monitored in the gas phase

Abstract

The enzymatic methylation of foreign sulphhydryl compounds in the microsomal fraction of mammalian liver was first reported by Bremer and Greenberg [2]. A broad degree of acceptor specificity was established, although "physiological" sulphhydryl-containing substances such as cysteine were inactive. On the other hand, structural requirements for the methyl donor molecule, S-adenosyl methionine, have been found to be quite specific [1] such that even close analogues are inactive, or at best inhibitory. A preliminary report from our laboratory has established the methylating capability of mammalian liver microsomes towards alkane thiols [4]. In contrast to the acceptors examined in earlier work, low members of the alkane thiol series are of endogenous physiological importance [3]. Methylation, which leads to blocking of the reactive sulphhydryl group can be considered as a detoxification process, and the activities observed in three independent studies on microsomes probably derive from a single enzyme or group of enzymes categorised by E.C. 2.1.1.9, S-adenosyl-L-methionine : thiol-S-methyltransferase. Straight-chain alkane thiols (C1-C10) and branchedchain isomers (C3-C~), and S-adenosyl methionine were purchased from commercial sources. The methyl thioether derivatives, required as reference samples, were synthesised from the respective alkyl bromides in reaction with sodium methyl thiolate. The microsomal fraction of mammalian liver homogenates was prepared by standard differential centrifugation techniques. The novelty of the present study lies partly in the method of assay of the methylation reaction, which in contrast to previous methods [1,2] does not require radioactivelylabelled substances. Incubations were carried out in sealed vials of a total volume 7 ml. The aqueous phase (1 ml) contained 1 mg of microsomal protein and 1 lamole of S-adenosyl methionine in 100 retool/1 of tris/HC1 buffer, pH 8. The system was thermostatted at 25 ~ C, and incubation was initiated by injection of the required amount of thin through the septum of the vial. Analysis was performed under the conditions reported previously [4] with removal of 5 gl of the gas phase in a gas-tight syringe and direct injection into the gas-chromatograph. Decrease in substrate and increase in product as a function of time could be monitored simultaneously as shown for ethane thiol in Fig. I, and integrated peak areas were converted into absolute quantities from calibration curves. The inset in the figure shows the kinetics ofmethylation for four different initial amounts of ethane thiol (So), whereby the gas-chromatograms in the figure represent one run. The degree of product formation, expressed as the amount of product at a given time, Pt, divided by the sum of product and residual substrate, (P, + S0, is plotted as a function of time, t. After an initial induction period, the plots were linear for at least 2 h. The quantity Pt/(l~t-Jr-St) is used rather than Pt directly to nullify errors arising from varying injected volumes from sample to sample. The slope of the plot, m, is thus equal to Pt/(Pt + St)" t. In the early stages of the reaction, Pt ~ St, so that 5', = So. Thus,