Abstract
This chapter discusses the formation of thymidine diphosphate (TDP)-4-acetamido-4,6-dideoxyhexoses from TDP-D-glucose. The assay procedure involves incubation of 14 C-TDP-D-glucose uniformly labeled in glucose with cell extracts of bacteria in the presence of Tris-HCl buffer, L-glutamate, pyridoxal phosphate, and acetyl CoA. The products and intermediates of the reactions are identified by paper chromatography of the nucleotide sugars and of the sugars obtained by acid or enzymatic hydrolysis of the nucleotides. The identification of the nucleotides and of the sugars liberated by mild acid hydrolysis or by digestion with venom phosphodiesterase and Escherichia coli phosphomonoesterase may be obtained by paper chromatography. Cochromatography of the radioactive nucleotides or free sugars with authentic compounds may be useful for the first identification. The biosynthetic reactions of TDP-4-acetamido-4,6-dideoxyhexoses in E. coli strains are also considered. Incubation of TDP-4-keto-6-deoxy- D -glucose with enzyme and L-glutamate, in the presence of pyridoxal phosphate, results in the disappearance of TDP-4-keto-6-deoxy-D-glucose, and the simultaneous formation of TDP-4-amino-4,6-dideoxy- D -glucose or -D-galactose. A 28-fold purification of transaminase from E. coli strain B is also described in the chapter.
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