Orientation and role of nucleosidediphosphatase and 5'-nucleotidase in Golgi vesicles from rat liver.
The fate of UDP formed during the galactosylation of added N-acetylglucosamine in Golgi vesicles isolated from rat liver using D2O-sucrose gradients has been determined. UDP-Gal labeled with [14C]uracil was used, and the products of the reaction were separated and quantitated by using high-pressure liquid chromatography. [14C]Uridine rather than [14C]UDP or [14C]UMP was found to accumulate, indicating the presence of both UDPase and UMPase activities in the Golgi. Golgi vesicles were shown to contain a nucleosidediphosphatase activity that is membrane bound. It appears to be located on the luminal face of the Golgi since it is activated 3-5-fold by detergents and 4-fold by treatment of the vesicles with Filipin. We have shown previously that Filipin disrupts the Golgi but does not solubilize membrane-bound enzymes. The nucleosidediphosphatase of the Golgi differs from that present in rough endoplasmic reticulum in its absolute requirement for Ca2+ for activity and in its substrate specificity that is higher for UDP than for IDP. Golgi vesicles also contain UMPase activity that is stimulated only 2-fold by detergents or Filipin. Concanavalin A inhibits this activity about 80% in both intact and detergent-treated vesicles. The Golgi UMPase is thus probably identical with 5'-nucleotidase. These results are consistent with histochemical evidence from other laboratories that indicate that 5'-nucleotidase is present on both sides of liver Golgi membranes. In the presence of concanavalin A and N-acetylglucosamine, intact Golgi vesicles were found to convert UDP-Gal to UMP. These findings indicate that UDP formed by galactosyltransferase in the lumen of the vesicles is rapidly converted to UMP by UDPase in the lumen but that UMP moves rapidly out of the lumen of the Golgi and is broken down to uridine by 5'-nucleotidase on the cytoplasmic side of the vesicles.
D015720:5'-Nucleotidase; D017766:Acid Anhydride Hydrolases; D000818:Animals; D002413:Cations, Divalent; D003208:Concanavalin A / Q000494:pharmacology; D005700:Galactosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009708:Nucleotidases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D002413:Cations, Divalent; D003208:Concanavalin A; D005700:Galactosyltransferases; D009708:Nucleotidases; D010744:Phosphoric Monoester Hydrolases; D015720:5'-Nucleotidase; D017766:Acid Anhydride Hydrolases; C023837:nucleoside-diphosphatase