| A unique phenol sulfotransferase (PST) isozyme, minoxidil ST (Mx-ST), catalyzes the conversion of Mx to Mx-sulfate (Mx-S). Mx-S has been determined to be the active agent in the vasodilatory and hair growth stimulatory effects of the drug. Mx-ST has been purified approximately 240-fold from male rat liver cytosol to apparent homogeneity by anion-exchange, hydroxylapatite, and ATP-agarose affinity chromatography. Purified Mx-ST is capable of sulfating Mx, a pyrimidine N-oxide, as well as the phenolic substrate, 4-nitrophenol (PNP). The apparent Kms of Mx-ST for Mx, PAPS, and PNP were 625 {dollar}mu{dollar}M, 5.0 {dollar}mu{dollar}M, and 0.5 {dollar}mu{dollar}M, respectively. Mx-ST displayed substrate inhibition at concentrations above 1.2 {dollar}mu{dollar}M PNP and 1 mM Mx.; Mx-ST exists as a homodimer of two 35 kDa subunits in its active form. Polyclonal antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35 kDa as determined by immunoblotting. Furthermore, this antibody has been used in immunolocalization studies of rat anagen pelage and vibrissae follicles. Localization of the enzyme in the hair follicle suggests that the outer root sheath may be an active site of Mx sulfation and in some way mediates its hair growth effects.; A cDNA encoding Mx-ST has been isolated from a {dollar}lambda{dollar}gtll cDNA library constructed from poly (A)+ RNA prepared from female Sprague-Dawley rat liver. The largest cDNA, designated Mx-STb, consists of 1245 base pairs and contains an open reading frame of 291 amino acids. The size of the protein obtained by in vitro translation of Mx-STb is identical to that of the pure protein. Results of initial studies of the expression of Mx-STb in COS-1 cells indicate that the expressed protein displays characteristic Mx-ST and PNP-ST activity, is recognized by rabbit polyclonal antibodies raised against pure rate liver Mx-ST, and migrates at approximately 35,000 daltons during SDS-PAGE.; Mx-ST is most likely the homologous enzyme to human liver phenol ST, P-PST. Antibodies raised to rat liver Mx-ST and human PST cross-react with purified P-PST and Mx-ST, respectively, suggesting the enzymes are structurally related. In addition, Mx-ST and P-PST exhibit similar substrate specificities. Finally, a high percentage of nucleotide and amino acid sequence identity (80%) is observed between these two enzymes. (Abstract shortened with permission of author.)... |
