Sulphotransferase activity has been assayed in porcine liver and testis cytosol using
either 3'-phosphoadenosine-5'-phospho [35S]sulphate (PAPS) or unlabelled PAPS as sulphate
donors. In porcine liver the sulphotransferase for DHA was linear for up to 10 min,
the optimum pH was 7.7 and optimum temperature, 37 degrees C. The apparent Km value
was found to be 91 mumol/l and the activity was inhibited non-competitively by 5 alpha-androst-16-en-3
beta-yl sulphate, with all concentrations used (0.02-25 mumol/l) inhibiting the enzyme
to the same extent. Time courses for sulphoconjugation of pregnenolone and 5 alpha-androst-16-en-3
beta-ol were linear for up to at least 10 min or up to only 5 min, respectively. The
optimum pH values and temperatures were pH 8.0 and 37 degrees C in each case. The
porcine testicular sulphotransferase activity for DHA as substrate was linear with
time up to 10 min, the apparent Km for the reaction was 2 mumol/l and apparent Vmax
10 nmol/l/mg/min. 5 alpha-Androst-16-en-3 beta-yl sulphate (11.3-45.2 mumol/l) failed
to inhibit the enzyme activity. The time-course for the reaction, when pregnenolone
was used as substrate, was also linear up to 10 min at the optimum pH 8.0 but, in
contrast to the reaction when DHA was the substrate, had an apparent Km of 20 mumol/l
and was inhibited by pregnenolone sulphate, 5 alpha-androst-16-en-3 beta-yl sulphate,
DHA and 5 alpha-androst-16-en-3 beta-ol, but not by DHA sulphate. 5 alpha-Androst-16-en-3
beta-yl sulphate inhibited the reaction non-competitively and to the same extent at
concentrations over the range 11.3-45.2 mumol/l. These data suggest that DHA and pregnenolone
may not be sulphoconjugated by the same sulphotransferase. With 5 alpha-androst-16-en-3
beta-ol as substrate, the time-course for its sulphate formation was linear up to
15 min, and this reaction could explain the quantities of 5 alpha-androst-16-en-3
beta-yl sulphate that are found endogenously in porcine testis. Our results further
suggest that these quantities could well inhibit the sulphation of pregnenolone in
porcine testis in vivo, and the possibility of control of sulphoconjugation in this
tissue is discussed. Having regard to the smaller quantities of 5 alpha-androst-16-en-3
beta-yl sulphate present in porcine liver, our results suggest that the sulphation
of DHA there may not be so much affected.