Nuleotide sugar photoaffinity analogs have proven to be useful in the identification and characterization of glycosyltransferases. A radioenzymatic synthesis of [32P]5-azido-UDP-N-acetylglucosamine has been accomplished using 5-azido-UTP, [gamma-32P]ATP, porcine N-acetylgalactosamine kinase, and Escherichia coli UDP-N-acetylglucosamine pyrophosphorylase, GlmU. This general enzymatic scheme was useful for the synthesis of [32P]5-azido-UDP-N-acetylgalactosamine and high-specific-activity [3H] or [32P]UDP-N-acetylhexosamines. A new chemical synthesis method for generating 5-azido-uridine compounds was also developed. [32P]5-Azido-UDP-N-acetylglucosamine was functionally characterized using different soluble and membrane-associated glycosyltransferases which utilize UDP-GlcNAc as a substrate. Site-specific photoincorporation was observed for partially purified GlmU and porcine UDP-GlcNAc pyrophosphorylase. The photoprobe also effectively photoincorporated into the alpha- and beta-subunits of purified bovine UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. Lastly, the photoprobe was also effective at photolabeling Streptococcus pyogenes hyaluronate synthase in membrane preparations.
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