Synthesis of uridine diphosphate glucose pyrophosphorylase during the development of Dictyostelium discoideum.

Uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9) from Dictyostelium discoideum has been purified to apparent physical and immunochemical homogeneity. The molecular weight estimates from gel diffusion and equilibrium sedimentation analyses are 390,000 and 384,000, respectively. The enzyme is polymeric apparently composed of a single monomeric species with a molecular weight of 55,000 as shown by dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity is 48,000 units per mg. The turnover numbers are 16,500 moles of UDP-glucose and 18,700 moles of glucose-l-P per min per mole of enzyme. The following Km values were obtained: glucose-l-P, 2.6 x 10m4 M; UTP, 1.1 X lo-’ M; UDP-glucose, 1.7 X lo-* M; pyrophosphate, 4.4 X lo-* M. UTP inhibits pyrophosphorylase activity both as a substrate at excess concentration and as a product, the latter inhibition being competitive with UDP-glucose. Pyrophosphate is a potent product inhibitor, not competitive with either glucose-l-P or UTP. Mixtures of the purified enzyme with crude extracts of cells harvested at different stages of fruiting body construction demonstrated that the assay of enzyme activity employed is an accurate reflection of the concentration of enzyme in the extracts. A high titer antiserum has been obtained which yields a single band in double diffusion assays with either purified enzyme preparations or crude extracts. Quantitative complement fixation and immune precipitation assays revealed no serological differences between the basal enzyme found in the vegetative cells and that which accumulates during fruiting body construction and demonstrated that the IOfold increase in the specific activity of the enzyme which occurs during specific stages of fruit construction is correlated with a proportionate increase in a single antigenic component. The enzyme was also immune precipitated from crude extracts of cells labeled with (35S]methionine during the period of enzyme accumulation. Polyacrylamide gel electrophoresis of the enzyme antibody complex solubilized with dodecyl sulfate showed a single labeled peak at the position