Isolation, partial purification, and characterization of a novel petromyzonol sulfotransferase from petromyzon marinus, Lamprey (Larval) liver

K. V. Venkatachalam, Domingo E. Llanos, Kristophe J. Karami, and Vladimir A. Malinovskii

Department of Biochemistry and Health Profession Division, Nova Southeastern University, Ft. Lauderdale, FL 33328-2018

Corresponding Author: venk{at}nova.edu

We have isolated, partially purified and characterized the 5 $alpha$ petromyzonol (5 $alpha$ -PZ), (5 $alpha$ -cholan- 3 $alpha$ , 7 $alpha$ , 12 $alpha$ , 24-tetrahydroxy-) sulfotransferase (PZ-SULT) from larval lamprey liver. Crude homogenates of liver extracts exhibited a PZ-SULT activity of 0.9120 pmol/min/mg in juvenile and 12.62 pmol/min/mg in larvae. Using concentrated crude larval liver extracts and 5 $beta$ -cholan substrates like cholic acid and its various derivatives; there was only background level of sulfonated products. The extracts were then tested for sulfotransferase activity using 5 $alpha$ -cholan substrates, 5 $alpha$ -PZ and 3-keto-5 $alpha$ -PZ which exhibited an activity of 231.5 pmol/min/mg and 180.8 pmol/min/mg respectively. With allocholic acid there was negligible sulfotransferase activity. This established that the sulfotransferase present in the lamprey larval liver extracts prefers (5 $alpha$ ) substrates and it is selective for hydroxyl at the C-24, for sulfonation. Partially purified PZ-SULT exhibits a pH optimum of 8.0; a temperature optimum of 22⁰C and the stability of the activity was linear for one hour. Using optimal conditions, PZ-SULT activity was then purified by DEAE ion exchange, gel filtration and PAP affinity column chromatography. PZ-SULT exhibited a Km of 2.5 $mu$ M for PAPS and a Km of 8 $mu$ M for PZ. The affinity purified peak PZ-SULT fraction exhibited a specific activity of 2038 pmol/min/mg. The peak activity fraction while subjected to SDS-PAGE, correlated to a protein with a molecular weight of 47 kDa. Photoaffinity labeling with PAP³⁵S cosubstrate, 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 of (M)SISQAVDAAFXEI, which possessed an overall ~ 35-40% homology with mammalian SULT2B1a.

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