PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING OF A NOVEL RAT-LIVER DOPA TYROSINE SULFOTRANSFERASE/

A novel sulfotransferase was purified from the rat liver cytosol to el ectrophoretic homogeneity via five column chromatography steps (hydrox ylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP- agarose II). The minimum molecular weight of the purified enzyme was d etermined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be similar to 33,000. Gel filtration chromatography revealed a nat ive molecular weight of similar to 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displaye d enzymatic activities, with a pH optimum of 9.25, toward various tyro sine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-ty rosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent K-m value of the enzyme (des ignated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 mu M of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N-te rminal sequencing, Three internal partial amino acid sequences, obtain ed by analyzing its proteolytic fragments, were found to be distinct f rom the homologous sequences of other known rat liver sulfotransferase s. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfec tion of COS-7 cells with an expression vector (pMSG . CMV) harboring t he full-length cDNA, a 33-kDa protein displaying enzymatic and immunol ogical properties similar to those of the purified Dopa/tyrosine sulfo transferase was expressed.