Purification and characterization of N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver.
N-Hydroxy-2-acetylaminofluorene (N-OH-2-AAF) sulfotransferase is an enzyme that catalyzes the sulfate transfer from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to N-OH-2-AAF to form a highly reactive product acetylaminofluorene N-sulfate. It has been purified about 2000-fold with a yield of over 12% from adult Sprague-Dawley male rat livers by an eight-step procedure. The final preparation was homogeneous on analytrical disc gel electrophoresis. The purified enzyme had activity toward p-nitrophenol with an approximately 1600-fold increase in specific activity over the crude homogenate, but it had almost no detectable activity toward steroids such as estrone, beta-estradiol, testosterone, dehydroisoandrosterone, and corticosterone. There was also very little sulfation activity toward serotonin and L-tyrosine methyl ester. The optimal pH for the enzyme activity is approximately 6.3 when measured in sodium phosphate buffer. Mg2+ at 6 to 9 mM could increase the enzyme activity up to 30%. Mn2+ activated the enzyme only slightly at very low concentrations. Zn2+, Co2+, Cu2+, and Ni2+ were all strongly inhibitory, but Ca2+ had very little effect. Thiol compounds were found to have a stabilizing effect and thiol-blocking reagents were potent inhibitors for this enzyme. The pure enzyme was very unstable especially in diluet solutions. The isoelectric point (pl) of the enzyme is 5.66 +/- 0.07. The molecular weight of the native enzyme was 68,000 +/- 500 as estimated by Sephadex G-100 and G-200 gel filtrations. A single component with molecular weight of 38,250 +/- 1,350 was observed on sodium dodecyl sulfate gel electrophoresis in the absence and presence of 2-mercaptoethanol. Comparison of the enzyme activity in mail and female rat livers at each stage of purification revealed that there was only a trace amount of N-OH-2-AAF sulfotransferase present in the female rat liver.
D000818:Animals; D002413:Cations, Divalent; D005260:Female; D005449:Fluorenes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006881:Hydroxyacetylaminofluorene / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats; D012737:Sex Factors; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D005449:Fluorenes; D006881:Hydroxyacetylaminofluorene; D013466:Sulfurtransferases; C021378:N-hydroxy-2-acetylaminofluorene sulfotransferase; D015238:Sulfotransferases