Isolation of tyrosylprotein sulfotransferase from rat liver.
1. Tyrosylprotein sulfotransferase (TPST) is involved in the posttranslational modification of proteins and plays a critical role in the biological activity and secretion of proteins. A simple method has been developed to isolate the TPST (28% yield) from rat liver, using polyclonal anti-TPST antibodies. 2. The protein fractions eluted from antibody affinity column showed TPST activity and revealed a 50-54 kDa protein band in the silver stained SDS-polyacrylamide gels. 3. The enzyme exhibited optimum activity at pH 5.5 with 20 mM MnCl2. Unlike the TPST activity of the Golgi membrane, the activity of the purified enzyme was not stimulated by NaF, 5'-AMP, and Triton X-100. 4. The antibody was also used to study the TPST protein turnover in rat liver of animals that were given [35S]methionine. The TPST protein synthesis assessed by measuring initial rates of incorporation of [35S]methionine into TPST protein showed enzyme synthesis for up to 60 min. 35S-labeled TPST protein of rat liver was degraded with a half-life of 30 hr. 5. The immunoaffinity purification method using rat liver as an enzyme source appeared to be very simple, rapid, and easy to perform with significant enzyme recovery. Further, the antibody was also found to be useful in the study involving TPST protein metabolism.
Ramaprasad
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P
Dental Research Center, UMDNJ-NJ Dental School, University of Medicine and Dentistry of New Jersey, Newark 07103-2400, USA.
Kasinathan
C
C
D000818:Animals; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015238:Sulfotransferases / Q000096:biosynthesis / Q000302:isolation & purification*
D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase