Isolation, partial purification, and characterization of a novel petromyzonol sulfotransferase from Petromyzon marinus (lamprey) larval liver.
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 beta-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 degrees C, and activity was linear for 1h. PZ-SULT exhibited a K(m) of 2.5 microM for PAPS and a K(m) of 8 microM 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 PAP(35)S, 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 (approximately 35-40%) homology with mammalian SULT2B1a.
Venkatachalam
K V
KV
Department of Biochemistry, College of Medical Sciences, Health Profession Division, Nova Southeastern University, Ft. Lauderdale, FL 33328-2018, USA. venk@nova.edu
Llanos
Domingo E
DE
Karami
Kristophe J
KJ
Malinovskii
Vladimir A
VA
D000595:Amino Acid Sequence; D000818:Animals; D007700:Kinetics; D007798:Lampreys / Q000254:growth & development; D007814:Larva / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008110:Liver Extracts / Q000737:chemistry; D013045:Species Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
D008110:Liver Extracts; D015238:Sulfotransferases