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 was purified from the Golgi membranes of rat submandibular salivary glands using a Cibacron blue F3GA affinity column chromatography. Antibodies raised in rabbit against TPST detected the purified enzyme (50-54 kDa) and proteins consisting of molecular mass 50-54 kDa in the Golgi membranes of liver, submandibular salivary glands, stomach, cerebellum, thalamus, and pituitary. The protein levels in liver and salivary glands were higher compared to those found in the stomach, cerebellum, thalamus, and pituitary. The levels of immunoreactivity in cytosol and endoplasmic reticulum fractions of salivary glands were either undetectable or very low. The antibody was also used to immunoprecipitate the TPST activity and to isolate protein by immunoaffinity column. MnCl2 was required for the purified TPST. The enzyme exhibited optimum activity between pH 6.2 and 6.8 at 20 mM MnCl2. The apparent K(m) values of the purified enzyme for poly-(Glu6, Ala3, Tyr1) (EAY: M(r) 47,000) and 3'-phosphoadenosine 5'-phosphosulfate were 3 and 20 microM, respectively. The results presented here collectively demonstrate the purification of TPST and, for the first time, development of polyclonal antibody that recognizes this enzyme.
William
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S
Dental Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark 07103-2400, USA.
Ramaprasad
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P
Kasinathan
C
C
D000818:Animals; D000906:Antibodies; D002846:Chromatography, Affinity; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008297:Male; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013363:Submandibular Gland / Q000201:enzymology*; D015238:Sulfotransferases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D014018:Tissue Distribution
D000906:Antibodies; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase