Purification and characterization of rat liver minoxidil sulphotransferase.
Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.
Hirshey
S J
SJ
Department of Pharmacology and Cancer Center, University of Rochester, NY 14642.
Falany
C N
CN
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002845:Chromatography; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008914:Minoxidil / Q000378:metabolism*; D009596:Nitrophenols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors; D015238:Sulfotransferases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D009596:Nitrophenols; D008914:Minoxidil; D000255:Adenosine Triphosphate; D015238:Sulfotransferases; C024836:4-nitrophenol