Effect of a fatty acid moiety of phospholipid and ceramide on purified GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) activity from embryonic chicken brain.
Galactosyltransferase, GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) has been characterized and solubilized from 19-day-old embryonic chicken brain, and purified to over 2000-fold using mixed-modal chromatography on a omega-aminohexyl Sepharose column and affinity chromatography on a UDP-hexanolamine Sepharose column. The activity of purified GalT-3 was modulated by phospholipids in vitro with stimulation observed specifically with dipalmitoyl phosphatidylethanolamine (PE). All natural phospholipids tested (PE, PC and PI) inhibited GalT-3 activity. Enzyme activity was affected by the structure of the phospholipid vesicle. It was stabilized by the hexagonal (dipalmitoyl PE) structure and inhibited by the bilayer (dielaidoyl PE) structure. The long-chain fatty acid moiety of the glycosphingolipid substrate, GM2, was found to be necessary for optimum enzyme activity. In the absence of fatty acid, the modified substrates, lyso-GM2 and acetyl-GM2, had a 10-fold increased Km and a 4-8 fold decreased Vmax compared to the normal substrate. We postulate that GalT-3 belongs to a group of glycosyltransferases having recognition for both the carbohydrate as well as the hydrophobic domains (HY-CARS) of their substrates and that the fatty acid moiety of either the substrate (GM2) or a heterotropic effector (phospholipid) plays an important role in regulating the activity of this enzyme.
Ghosh
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S
Department of Chemistry & Biochemistry, University of Notre Dame, Indiana 46556.
Das
K K
KK
Daussin
F
F
Basu
S
S
D000818:Animals; D001921:Brain / Q000196:embryology / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D002642:Chick Embryo; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D005227:Fatty Acids / Q000378:metabolism; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051147:Ganglioside Galactosyltransferase; D007700:Kinetics; D008081:Liposomes; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*
D002518:Ceramides; D005227:Fatty Acids; D008081:Liposomes; D010743:Phospholipids; D005700:Galactosyltransferases; D051147:Ganglioside Galactosyltransferase