Physical characterization of a monoamine-sulfating form of phenol sulfotransferase from human platelets.
The purification to homogeneity and physical characterization of a monoamine-sulfating form of phenol sulfotransferase (PST) from human platelets is described. DEAE-cellulose chromatography of a 100,000 x g supernatant solution of homogenized human platelets revealed the presence of two peaks of both dopamine- and phenol-sulfating activity, termed M- and P-PST, respectively. The latter dopamine-sulfating form eluting from the ion exchange column, MII-PST, was purified approximately 10,000-fold to electrophoretic homogeneity by Sephacryl S-200 HR and 3'-phosphoadenosine-5'-phosphate-agarose chromatography. The final specific activity of the enzyme was 930 nmol/min/mg of protein. As determined by the hydrodynamic properties of MII-PST, the native Mr was approximately 69,000. The frictional ratio (f/fo) was estimated to be 1.28, indicating that the enzyme possesses a relatively low degree of asymmetry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the affinity-purified enzyme revealed the presence of single Mr species of approximately 34,000, suggesting that MII-PST exists as a homodimer in vivo. Isoelectric focusing of purified MII-PST yielded a single protein species with a pl of 4.7. The sulfhydryl-modifying reagent N-ethylmaleimide (50 microM) was found to inactivate MII-PST in a time-dependent manner. This inactivation was totally prevented by saturating concentrations of 3'-phosphoadenosine-5'-phosphosulfate, whereas dopamine bestowed only partial protection to the enzyme. These results suggest that at least one sulfhydryl moiety is present at the active site of MII-PST.
Heroux
J A
JA
Department of Pharmacology and Therapeutics, State University of New York at Buffalo, School of Medicine and Biomedical Sciences 14214.
Roth
J A
JA
D000588:Amines / Q000097:blood*; D015239:Arylsulfotransferase; D001792:Blood Platelets / Q000201:enzymology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D004298:Dopamine / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D013431:Sulfates; D013466:Sulfurtransferases / Q000037:antagonists & inhibitors / Q000097:blood*
D000588:Amines; D046911:Macromolecular Substances; D013431:Sulfates; D013466:Sulfurtransferases; D015239:Arylsulfotransferase; D004298:Dopamine