PURIFICATION OF TYROSYLPROTEIN SULFOTRANSFERASE FROM RAT SUBMANDIBULAR SALIVARY-GLANDS

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands. In the present study, this enzyme w as purified from the Golgi membranes of rat submandibular salivary gla nds using a Cibacron blue F3GA affinity column chromatography. Antibod ies raised in rabbit against TPST detected the purified enzyme (50-54 kDa) and proteins consisting of molecular mass 50-54 kDa in the Gels m embranes of liver, submandibular salivary glands, stomach, cerebellum, thalamus, and pituitary. The protein levels in liver and salivary gla nds were higher compared to those found in the stomach, cerebellum, th alamus, and pituitary. The levels of immunoreactivity in cytosol and e ndoplasmic reticulum fractions of salivary glands were either undetect able or very low. The antibody was also used to immunoprecipitate the TPST activity and to isolate protein by immunoaffinity column. MnCl2 w as required for the purified TPST. The enzyme exhibited optimum activi ty between pH 6.2 and 6.8 at 20 mM MnCl2. The apparent K-m values of t he purified enzyme for poly-(Glu6, Ala3, Tyr1) (EAY: M(r) 47,000) and 3'-phosphoadenosine 5'-phosphosulfate were 3 and 20 mu M, respectively . The results presented here collectively demonstrate the purification of TPST and, for the first time, development of polyclonal antibody t hat recognizes this enzyme. (C) 1997 Academic Press.