Thiol-dependent changes in the properties of rat liver sulphotransferases

Abstract

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The K(m) values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5mum and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0 degrees C when the K(m) and V(max.) values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0 degrees C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The K(m) value for adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.

Studies on the sulphation of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds by rat tissues

“…,I_ (nmol of 35S/h per mg of protein) 3-0-Sulphate 4-0-Sulphate 29.7 62.5 37.5 1.7 support. The finding that L-dopa itself does not apparently undergo any sulpho-conjugation, whereas dopamine readily does so, is not altogether surprising since a similar situation obtains with tyrosine and tyramine in the sulphating system of the liver cytosol (Segal & Mologne, 1959;Barford & Jones, 1971a). These observations add further support to the conclusions that the enzyme responsible for all these sulpho-conjugations is the L-tyrosine methyl ester sulphotransferase, first described by Mattock & Jones (1970).…”

“…Determination of sulphate ester formation. The composition ofthe reaction mixture was based on that used by Barford & Jones (1971a) and consisted of 10,ul of enzyme preparation, 50,u of 0.3mM-adenosine 3'-phosphate 5'-[35S]sulphatophosphate, 100,ul of 0.1 M-Tris-HCl buffer, pH7.5, containing the acceptor substrate and in some cases 0.48,umol of dithiothreitol. All reaction mixtures were incubated for 30min at 37°C and the reaction was terminated by placing the tubes in boiling water for 1 min.…”

“…Several minor radioactive components, one of them having a mobility coincident with that of the authentic 4-0-sulphate isomer, were detectable on chromatograms but these were present in small amounts only relative to the major product (less than 1 %). A comparison of the activity of the enzyme preparation towards dopamine and p-nitrophenol, a recognized substrate for phenolsulphotransferase (Mattock & Jones, 1970;Barford & Jones, 1971a), showed that the former is the better sulphate acceptor. The presence of 3 mM-dithiothreitol in the incubation mixture increased the amount ofproduct formed from dopamine by 134%, whereas the effect on the formation of p-nitrophenyl sulphate when p-nitrophenol was the acceptor was very much less (35 Y.).…”

Purification and properties of a phenol sulphotransferase from Euglena using l-tyrosine as substrate

“…In general, the usual phenol sulphotransferases from mammalian sources appear to accept a much broader spectrum of substrates than the Euglena enzyme. It is of interest in this connection that the mammalian enzymes that use L-tyrosine methyl ester have rather high Km values for this substrate (2900-5000 ,M) (Banerjee and Roy, 1968;Barford and Jones, 1971) compared with the Euglena enzyme ( Table 2). The usual mammalian phenol sulphotransferases appear to have evolved as enzymes of detoxification, and evolutionary selection would favour a wide range of substrate acceptance.…”

“…PAPS:phenol sulphotransferases or arylsulphotransferases (EC 2.8.2.1) active in detoxification (see Jakoby et al, 1980;Roy, 1981;Ramaswamy and Jakoby, 1987) generally have rather broad specificities for the phenols they will use as sulphate acceptors. Although in some cases tyrosine methyl ester is used as a substrate (Banerjee and Roy, 1968; Barford and Jones, 1971), activity is not found with free tyrosine (Jakoby et al, 1980). For this reason the nature of the system in Euglena organelles forming Tyr(SO3H) from tyrosine and PAPS is of interest and led us to purify the enzyme and7 determine its properties.…”