Purification of a rat liver phenol sulphotransferase (P-STG) with the aid of guanidine hydrochloride treatment.
An isoenzyme of phenol sulphotransferase, designated P-STG, was purified 157-fold from male rat liver cytosol by diethylaminoethyl-cellulose (DEAE-cellulose) and agarose-hexane-adenosine-3',5'-bisphosphate affinity chromatography. The P-STG fraction obtained after DEAE-cellulose chromatography rapidly lost its activity during storage at 4 degrees C, however, the activity was recovered by the addition of 1.6 M guanidine hydrochloride (Gndn HCl) followed by dialysis. Gndn HCl also substantially improved the yield of P-STG in a subsequent purification step using affinity chromatography, while the specific activity of the purified P-STG was not changed by Gndn HCl treatment. It is possible that the Gndn HCl treatment caused P-STG recovery from an inactivated to an active form rather than reactivating it for increased activity. Purified P-STG is a homodimer with a native molecular mass of 67 kDa; the subunit molecular mass is 35 kDa. Immunoblot analysis carried out with antibodies raised against the purified enzyme indicated that male rat liver contains a higher level of the enzyme than female rat liver. This enzyme is also expressed in the kidney and the stomach. P-STG reaches maximum activity when 1-naphthol, 2-naphthol and 4-nitrophenol are used as substrates at pH 5.5. Using dopamine as a substrate the pH optimum is about 9.0. P-STG activity is markedly inhibited by the addition of sodium chloride to the reaction mixture.
Homma
H
H
Kyoritsu College of Pharmacy, Tokyo, Japan.
Kamakura
M
M
Nakagome
I
I
Matsui
M
M
D000818:Animals; D015239:Arylsulfotransferase / Q000302:isolation & purification*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D006146:Guanidines / Q000494:pharmacology*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats
D006146:Guanidines; D007202:Indicators and Reagents; D007527:Isoenzymes; D015239:Arylsulfotransferase