Synthesis of a new photoaffinity probe, 5-azido-[32P]UDPxylose, by UDPglucuronate carboxylyase from wheat germ.

ABSTRACT

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.