Coupling of uridine-5'-diphosphate (UDP) formation and nicotinamide adenine dinucleotide (NAD+) reduction for cytochemical localization of glycosyltransferases.
A technique applicable to the cytochemical localization of glycosyltransferases through a series of coupled enzyme reactions is described. Uridine-5'-diphosphate (UDP) formed by glycosyltransferases is first phosphorylated to uridine-5'-triphosphate (UTP) by nucleoside 5'-diphosphate kinase. The UTP plus exogenous glucose-1-phosphate is converted into UDP-glucose by uridine-5'-diphosphoglucose pyrophosphorylase. UDP-glucose is then oxidized by uridine-5'-diphosphoglucose dehydrogenase to form UDP-glucuronic acid and reduced nicotinamide adenine dinucleotide (NADH). The NADH is utilized by membrane-located NADH-ferricyanide oxidoreductases in the presence of a copper salt to form electron-dense deposits of cupric ferrocyanide (Hatchett's brown). Using this technique, galactosyltransferase has been localized in cisternae (including the central midregions of the cisternae) of Golgi apparatus isolated from rat liver. Reactivity is absent from the cis-most cisternae and membrane elements. The reaction is dependent on UDP-galactose and inhibited by ethylene diaminetetraacetic acid and puromycin. the latter is a known inhibitor of galactosyltransferase of rat liver Golgi apparatus. The reaction is adaptable by varying the sugar nucleotide donor and acceptor to any glycosyltransferase utilizing UDP-sugars (except UDP-glucose). Presently it is restricted to isolated membrane fractions and permeabilized cells due to the need for accessibility of reagents and coupling enzymes.
D000818:Animals; D005700:Galactosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D006651:Histocytochemistry / Q000379:methods*; D008099:Liver / Q000166:cytology / Q000201:enzymology; D008297:Male; D009243:NAD / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014500:Uracil Nucleotides / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism*
D014500:Uracil Nucleotides; D009243:NAD; D014530:Uridine Diphosphate; D005700:Galactosyltransferases