Synthesis of a new photoaffinity probe, 5-azido-[32P]UDPxylose, by UDPglucuronate carboxylyase from wheat germ.
The enzyme, UDPglucuronic acid carboxylase (EC 4.1.1.35), was extensively purified from wheat germ, and was used to convert 5-azido-[32P]UDPglucuronic acid to 5-azido-[32P]UDPxylose, for use as a new photoaffinity probe. The carboxylyase was purified approximately 1200-fold using conventional methods, and the enzyme preparation, at the final stage of purification, was stable to storage at -20 degrees C for at least 9 months with little or no loss of activity. The partially purified carboxylyase catalyzed the conversion of 5-azido-[32P]UDPglucuronic acid to 5-azido-[32P]UDPxylose in good yield, and the UDPxylose probe was purified by ion-exchange chromatography, and characterized. The newly synthesized photoaffinity analog, 5-azido-[32P]UDPxylose, should be a valuable tool in the purification of various xylosyltransferases.
Kyossev
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ZN
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock.
Drake
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RR
Kyosseva
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Elbein
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AD
D000345:Affinity Labels / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000302:isolation & purification / Q000494:pharmacology*; D014908:Triticum; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism*
D000345:Affinity Labels; C071932:5-azido-UDP-glucuronic acid; D014535:Uridine Diphosphate Glucuronic Acid; D014540:Uridine Diphosphate Xylose; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase