Metabolism of uridine 5'-diphosphate-glucose in golgi vesicles from pea stems

Uridine 5'-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[3H]Glc, [3H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [alpha-32P]UDP-Glc, [beta-32P]UDP-Glc, and [3H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5'-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.

Topography and Function of Golgi Uridine-5[prime]-Diphosphatase from Pea Stems.

Golgi UDPase is an enzyme that has been shown to function in polysaccharide biosynthesis, but its role in this process is not yet clear. In this study we identified Golgi UDPase activity in pea (Pisum sativum) stems and differentiated it from another UDPase activity. We demonstrated that Golgi UDPase is an integral membrane protein, based on specific partitioning of this activity into Triton X-114. Analysis of its topology using sealed, right-side-out Golgi vesicles and treatment with proteinase K suggested that its active site faces the Golgi lumen. Studies aimed at understanding the function of Golgi UDPase by incubating Golgi vesicles with [beta]-32P]UDP-glucose (Glc) to generate [beta]-32P]UDP upon Glc transfer in situ showed that 32Pi, but not [beta]-32P]UDP, was formed, suggesting that UDPase quickly hydrolyzed the UDP formed during Glc polymerization. We found that the Golgi UDPase was highly active in the elongating region of the third internode, whereas no activity was detected in the first and second internodes of etiolated pea seedlings. These results suggest that UDPase removes the UDP formed during Glc polymerization and could be important in the mechanism of polysaccharide biosynthesis.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney.

ATP-diphosphohydrolase (apyrase. EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metals, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similarities in Mr. bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specific ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney

ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metais, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similaiities in Mr, bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specifjc ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.