Purification and characterization of human liver phenol-sulfating phenol sulfotransferase.
The phenol-sulfating form of phenol sulfotransferase (P-PST) was purified and characterized from human liver cytosol using DEAE-cellulose, Sephacryl S-200, and 3',5'-diphosphoadenosine-agarose affinity chromatography. During the purification procedure, P-PST was resolved from the monoamine-sulfating form of phenol sulfotransferase (M-PST) and dehydroepiandrosterone sulfotransferase, which are also present in human liver cytosol. P-PST activity was purified 560-fold as compared to liver cytosol and the purified enzyme possessed a specific activity of 340 nmol phenol sulfated per minute per milligram protein. Enzymatically active P-PST has an apparent molecular size of 68,000 Da as determined by Sephacryl S-200 chromatography and a subunit molecular weight of 32,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that P-PST exists in vivo as a homodimer. Antibodies raised to human platelet M-PST cross-reacted strongly with pure P-PST suggesting the two PSTs are structurally closely related. Two types of P-PST activity have been identified in different human livers by their thermostability and elution during anion-exchange chromatography. Each of the livers examined possessed only one type of P-PST activity. Both types of P-PST were shown to possess the same subunit molecular weight and immunoreactivity, whereas the differences in thermostability of the two P-PST activities appeared to be related to the method of preparation of liver cytosol. Both types of P-PST activity were inhibited to similar extents by incubation with 50 microM N-ethylmaleimide or 5 mM phenylglyoxal. These results suggest that the two types of P-PST in different human livers are very similar and probably represent different allelic forms of the enzyme.
Falany
C N
CN
Department of Pharmacology, University of Rochester, New York 14642.
Vazquez
M E
ME
Heroux
J A
JA
Roth
J A
JA
D015239:Arylsulfotransferase / Q000302:isolation & purification*; D001792:Blood Platelets / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010658:Phenylglyoxal / Q000494:pharmacology; D013379:Substrate Specificity
D015239:Arylsulfotransferase; D010658:Phenylglyoxal; D005033:Ethylmaleimide