Characterization and purification of fucosyltransferases from the cytosol of rat colon.
The occurrence and baseline characteristics of fucosyltransferases (alpha-1,2, alpha-1,3 and alpha-1,4) in the cytosol (soluble) and pellet (membrane-bound) of rat colon have been studied since the fucosylation process is known to alter in colon pathology. All enzymes studied in the colon pellet had higher activity when compared to the cytosol. The colon pellet alpha-1,3 fucosyltransferase preferred desialylated alpha 1-acid glycoprotein as acceptor substrate. Both soluble and membrane-bound enzymes, alpha-1,2 and alpha-1,3 fucosyltransferases, required Mn2+, Mg2+ and Ca2+ for maximum activity but were inactivated by Cu2+ ions. Both soluble alpha-1,2 and alpha-1,3 fucosyltransferases showed optimal activity at pH 6.0, whereas the optimum for their membrane-bound activities were at pH 5.8 and 6.2, respectively. Furthermore, a soluble alpha-1,3 fucosyltransferase from rat colon was purified and during purification the co-presence of alpha-1,3/4 fucosyltransferase was detected. The acceptor of preference for the purified soluble alpha-1,3 fucosyltransferase was desialylated glycoprotein while low molecular weight substrates were poor acceptors. Both the purified fucosyltransferases were inhibited by N-ethylmaleimide. The M(r) values determined by SDS-PAGE electrophoresis of alpha-1,3/4 fucosyltransferase and of alpha-1,3 fucosyltransferase were 68,780 and 40,680 respectively. In conclusion, based on their properties, the purified soluble colon alpha-1,3 fucosyltransferase appeared to be of plasma-type (or FT-I) while the soluble alpha-1,3/4 fucosyltransferase corresponded to Lewis-type or FT-III.
Karaivanova
V
V
Department of Biochemistry, Memorial University of Newfoundland, St. John's Canada.
Mookerjea
S
S
Hunt
D
D
Nagpurkar
A
A
D000818:Animals; D002240:Carbohydrate Sequence; D003106:Colon / Q000201:enzymology* / Q000648:ultrastructure; D003600:Cytosol / Q000201:enzymology*; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013379:Substrate Specificity
D008565:Membrane Proteins; D005647:Fucosyltransferases