Stimulation of tyrosylprotein sulfotransferase activity by ethanol: role of increased enzyme level.
Tyrosylprotein sulfotransferase (TPST), an enzyme involved in the posttranslational modification of proteins, plays important role in the biological activity and secretion of proteins. Previously we have shown an increased activity of this enzyme in gastric mucosa of alcoholics. In the present study, effect of ethanol on TPST was examined in rat liver and gastric mucosa utilizing enzyme assays and Western blot analyses for TPST levels. Male Sprague-Dawley rats were pair-fed Leiber-DeCarli liquid diets for 10 days and controls received a liquid diet in which dextrose was isocalorically substituted for ethanol. After ethanol feeding, rats were sacrificed and liver and gastric mucosa were processed for Golgi membrane preparation. The TPST activity was measured using poly(Glu6, Ala3, Tyr1) as the sulfate acceptor and PAPS as sulfate donor. There was a threefold increase in TPST activity of gastric mucosa of animals subjected to chronic alcohol feeding. In the liver, the increase in tyrosine sulfating activity was also around threefold. The kinetic studies performed to understand the mechanism involved in ethanol stimulation of TPST activity showed no change in the Km values of the enzyme by ethanol. In control and ethanol-treated animals, the Km for EAY was 0.41-0.53 and 0.43-0.53 microM, and the Km for PAPS was 10-12.5 and 9-17 microM, respectively. The Vmax in ethanol-fed animals was increased by 1.5- to 2-fold. The increase in TPST activity in experimental rats was further tested by analyzing the Western blots by Imaging Densitometer for TPST levels. Analysis of TPST levels also showed over threefold increase in the stomach and liver of ethanol-fed rats. Our results indicate that stimulation of TPST by ethanol involves increased TPST level rather than change in affinity for its substrates.
Kasinathan
C
C
Dental Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark 07103-2400, USA.
Ramaprasad
P
P
William
S
S
Espina
N
N
D000818:Animals; D015153:Blotting, Western; D003720:Densitometry / Q000379:methods; D000431:Ethanol / Q000494:pharmacology*; D005753:Gastric Mucosa / Q000201:enzymology; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D008565:Membrane Proteins; D013431:Sulfates; D000431:Ethanol; D014443:Tyrosine; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase