UDP-sugar pyrophosphatase of rat retina: subcellular localization and topography.

Rat retinal tissue possesses as a developmentally regulated, highly active pyrophosphatase activity that hydrolyzes UDP-GalNAc and UDP-Gal but not CMP-NeuAc (Martina et al.: J Neurochem 62:1274-1280, 1995). We show here that this activity, measured with UDP-[3H]GalNAc as substrate, is associated to the membrane fraction of rat retinal homogenates and, upon subfractionation by isopycnic centrifugation in sucrose density gradients, is concentrated in fractions enriched in light Golgi membranes. We examined also the topographic disposition of the catalytic site of the enzyme in the transverse plane of the membranes by measuring the effect of protease treatment and of added EDTA on its activity. Pronase inhibited 50% of the translocation of UDP-[3H]GalNAc to the lumen of the Golgi vesicles but did not affect the enzyme activity either in the absence or in the presence of detergent. EDTA, a membrane-impermeant molecule, inhibited 90% of the activity of the enzyme but did not affect translocation of UDP-[3H]GalNAc and inhibited only 25% the incorporation of [3H]GalNAc into endogenous glycoconjugates. These results indicate that the translocation of UDP-[3H]GalNAc was not necessary for hydrolysis to occur and strongly suggest that the catalytic site of the UDP-sugar pyrophosphatase is oriented toward the cytosolic side of the Golgi vesicles. We speculate that this activity limits the availability of UDP-GalNAc to its specific translocator and, consequently, the luminal concentration of the nucleotide in the Golgi vesicles. In this way, by limiting the availability of UDP-GalNAc for the conversion of GM3 to GM2 by the GM3:N-acetyl-galactosaminyl transferase, it would contribute to the preferential use of GM3 for synthesis of GD3 and other "b" pathway gangliosides that are characteristic of the rat retina.