Abstract
A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP-agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be approximately 33,000. Gel filtration chromatography revealed a native molecular weight of approximately 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent Km value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 microM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N-terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. 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 transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.
Highlights
A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps
It was noted that, during the elution from the DEAE Bio-Gel A anion exchange chromatography step, the Dopa/tyrosine sulfotransferase activity was well separated from the major phenol sulfotransferase activity
A second ATP-agarose affinity chromatography was used in the final step of purification to remove small amounts of contaminating proteins and, at the same time, improve the specific activity of the purified Dopa/tyrosine sulfotransferase by removing denatured enzyme molecules that had lost the PAPS binding activity
Summary
A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATPagarose II). Aiming at demonstrating the first of these two mechanisms, a great number of studies using various cell homogenates or purified aryl sulfotransferases have, persistently failed to reveal the enzymatic activity that catalyzes the sulfation of free L-tyrosine (1, 9 –12). Among the different mammalian aryl sulfotransferases characterized, only the rat liver type IV aryl sulfotransferase, named the “tyrosine-ester sulfotransferase,” can use tyrosine esters or N-terminally located tyrosine residues as substrates This enzyme, cannot catalyze the sulfation of unmodified L-tyrosine [13]. In view of the widespread occurrence of the post-translational tyrosine sulfation among proteins of multicellular eukaryotic organisms [14], it has become increasingly accepted that the free TyrS excreted in mammalian urine is probably derived exclusively from the degradation of tyrosine sulfated proteins [1, 6, 15,16,17]. Rat Liver Dopa/Tyrosine Sulfotransferase is truly the sole source of the free TyrS produced and released by mammalian animals
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