Isolation, partial purification, and characterization of a novel petromyzonol sulfotransferase from Petromyzon marinus (lamprey) larval liver

We have isolated, partially purified, and characterized the 5α-petromyzonol (5α-PZ), (5α-cholan- 3α, 7α, 12α, 24-tetrahydroxy-) sulfotransferase (PZ-SULT) from larval lamprey liver. Crude liver extracts exhibited a PZ-SULT activity of 0.9120 pmol/min/mg in juvenile and 12.62 pmol/min/mg in larvae. Using crude larval liver extracts and various 5 β-cholan substrates and allocholic acid there was negligible activity, however, with 5α-PZ and 3-keto-5α-PZ the SULT activity was 231.5 pmol/min/mg and 180.8 pmol/min/mg respectively. This established that the sulfotransferase of lamprey larval liver extracts prefers (5 α) substrates and it is selective for hydroxyl at C-24. PZ-SULT was purified through various chromatography procedures. Partially purified PZ-SULT exhibited a pH optimum of 8.0, a temperature optimum of 22°C, and activity was linear for 1h. PZ-SULT exhibited a Km of 2.5 μM for PAPS and a K m of 8 μM for PZ. The affinity purified peak PZ-SULT exhibited a specific activity of 2,038 pmol/min/mg. The peak protein upon SDS-PAGE, correlated to an Mw 47 kDa. Photoaffinity labeling with PAP35S, specifically crosslinked the 47 kDa protein, further confirming the identity of PZ-SULT. Partial amino acid sequencing of the putative 47 kDa PZ-SULT protein yielded a peptide sequence (M)SISQAVDAAFXEI, which possessed an overall (∼35-40%) homology with mammalian SULT2Bla.