J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M300346-JLR200 on December 1, 2003

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Journal of Lipid Research, Vol. 45, 486-495, March 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

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

K. V. Venkatachalam1,*, Domingo E. Llanos*, Kristophe J. Karami* and Vladimir A. Malinovskii{dagger}

* Department of Biochemistry, College of Medical Sciences, Health Profession Division, Nova Southeastern University, Ft. Lauderdale, FL 33328-2018
{dagger} University of Miami School of Medicine, Department of Biochemistry and Molecular Biology, Miami, FL 33101

1 To whom correspondence should be addressed. e-mail: 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 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 ß-cholan substrates and allocholic acid there was negligible activity, however, with 5{alpha}-PZ and 3-keto-5{alpha}-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 {alpha}) 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 Km 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 SULT2B1a.

Abbreviations: 3-keto-PZ, 3-keto-petromyzonol; PAP, 3'-phosphoadenosine 5'-phosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PZ, petromyzonol; PZS, petromyzonol sulfate; PZ-SULT, petromyzonol sulfotransferase; SULT, sulfotransferase

Supplementary key words petromyzonol sulfate • chemoattractant • pheromone


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