Biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer by a novel glucuronyltransferase (GlcAT-1) from embryonic chicken brain.
A novel glucuronyltransferase (GlcAT-1) has been detected in embryonic chicken brains. This enzyme catalyzes the biosynthesis in vitro of glucuronic acid containing glycolipids starting from neolactotetraosylceramide (nLcOse4Cer) and neolactohexaosylceramide (nLcOse6Cer). The activity is present primarily in the Golgi-rich membrane fraction and can be extracted (60%) from the membrane using a neutral detergent, Nonidet P-40, at pH 7.0. The detergent-solubilized GlcAT-1 is stable (70%) at -20 degrees C for at least 4 months. Both membrane-bound GlcAT-1 and solubilized GlcAT-1 show similar pH optima, 6.5-7.0, in HEPES buffer. The Km values were 15 and 200 microM with UDP-[14C] GlcA and nLcOse4Cer, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The purified 14C radioactive product that comigrated with chemically characterized GlcA beta 1-3nLcOse4Cer (GlcA-nLc4) also yielded a positive immunostain with monoclonal antibody (human IgM-RI). The anomeric linkage was established as beta-linked GlcA to the terminal galactose of the substrate, as evidenced by 90-99% cleavage of the terminal [14C] GlcA by purified Helix pomatia and limpet glucuronidases. Permethylation studies of the radioactive product obtained from [6-3H]Gal beta 1-4LcOse3Cer and non-radioactive UDP-GlcA showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed enzymatic product. These studies established the structure of the biosynthesized product from nLcOse4Cer as GlcA beta 1-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4Glc-ceramide.
Das
K K
KK
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Basu
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Basu
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Chou
D K
DK
Jungalwala
F B
FB
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D005915:Globosides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000096:biosynthesis* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D013045:Species Specificity; D014018:Tissue Distribution
D002413:Cations, Divalent; D005915:Globosides; C036363:paragloboside; C067806:glucuronyltransferase GlcAT-1; D014453:Glucuronosyltransferase