Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues.
The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.
Rens-Domiano
S
S
Department of Pharmacology and Therapeutics, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
Roth
J A
JA
D000818:Animals; D001921:Brain / Q000201:enzymology; D002413:Cations, Divalent; D003902:Detergents / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009928:Organ Specificity; D011499:Protein Processing, Post-Translational; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D015238:Sulfotransferases / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D003902:Detergents; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase