Sulfation of tert-butoxycarbonylcholecystokinin and other peptides by rat liver tyrosylprotein sulfotransferase

The sulfoconjugation of tyrosyl residues is a widespread posttranslational modification of biologically active proteins and peptides. The rat liver Golgi enzyme tyrosylprotein sulfotransferase has previously been shown to catalyze the transfer of a sulfate moiety from 5'-phosphoadenosine-3'-phosphosulfate to the synthetic acidic polymer poly(Glu₆, Ala₃, Tyr₁) (EAY). In this study, we further characterized the biochemical properties and the substrate specificity of rat liver tyrosylprotein sulfotransferase using a variety of synthetic peptides, including EAY, tert-butoxycarbonylcholecystokinin-8 (Boc-CCK), and two carboxy terminal peptide fragments of complement component C4. The data demonstrate that all substrates can be sulfated by an isolated Golgi membrane fraction. In addition, Boc-CCK was also found to be sulfated by a cytosolic sulfotransferase. Using an enriched Golgi fraction, rat liver tyrosylprotein sulfotransferase sulfation of Boc-CCK displayed a pH optimum between 6.0 and 6.5, was stimulated by 50-150 mM NaCl, and required either Mn²⁺ or Mg²⁺ for maximal activity. In contrast, EAY sulfation displayed a pH optimum of 6.7, was not stimulated by NaCl, and required Mn²⁺ for maximal activity. Both Boc-CCK and EAY sulfation were similarly decreased when the enriched Golgi preparation was preincubated at 45° for varying lengths of time. The particulate tyrosylprotein sulfotransferase could be effectively solubilized by 1% Triton X-100 in the presence of 0.2 M NaCl and anion exchange chromatography of the solubilized enzyme yielded a single peak of Boc-CCK- and EAY-sulfating activities. Rat liver tyrosylprotein sulfotransferase was also shown to sulfate a variety of other acidic and basic synthetic tyrosine-containing polymers, as well as two synthetic peptides of 10 and 16 amino acid residu corresponding to the C-terminal portion of C4.