Characterization of solubilized GlcAT-1 (UDP-GlcA: nLcOse4Cer beta 1-3 glucuronyltransferase) activity from embryonic chicken brain and its inhibition by D-erythro-sphingosine.
Glycolipid glucuronyltransferase activity (GlcAT-1) has been solubilized and characterized from 19-day-old embryonic chicken brain Golgi-rich membranes. The enzyme catalyzes the biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer glycolipid using neolactetraosylceramide (nLcOse4Cer, Gal beta 1-4GlcNAc beta 1-3Gal beta-1-4Glc-Cer) as the substrate. The membrane-bound enzyme shows optimum activity in the presence of neutral detergents such as Triton CF-54, Triton DF-12, and Nonidet P-40. Approximately 60% of the enzyme activity can be solubilized from the Golgi membrane by Nonidet P-40. The solubilized GlcAT-1 activity is inhibited by different salts such as NaCl, NaBr, NaI, and NaOAc, but not by sodium fluoride (up to 0.4 M concentration). Desialyzed alpha 1 acid glycoprotein (SA alpha 1AGP) can be used as a substrate for glucuronyltransferase. Competition studies between glycolipid (nLcOse4Cer) and glycoprotein SA alpha 1AGP) substrates show a mixed type of inhibition. Phospholipids, in particular phosphatidylglycerol, stimulate solubilized GlcAT-1 activity, while D-erythro-sphingosine, a metabolite of glycosphingolipids, is inhibitory (50% inhibition at 0.8 mM D-erythro-sph). These results demonstrate that both phospholipid as well as sphingosine might be involved in modulating glucuronyltransferase activity.
Das
K K
KK
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Basu
M
M
Li
Z X
ZX
Basu
S
S
Jungalwala
F
F
D000818:Animals; D001921:Brain / Q000196:embryology / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002642:Chick Embryo; D003902:Detergents; D005915:Globosides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D012995:Solubility; D013110:Sphingosine / Q000378:metabolism / Q000494:pharmacology*
D003902:Detergents; D005915:Globosides; D008565:Membrane Proteins; D010743:Phospholipids; C036363:paragloboside; C067806:glucuronyltransferase GlcAT-1; D014453:Glucuronosyltransferase; D013110:Sphingosine