Rabbit liver UDP-glucose pyrophosphorylase has been crystallized and further purified to a specific activity of 200 by using preparative centrifugation with sucrose density gradients. The pyrophosphorylase requires a divalent cation. Magnesium is preferred, although manganese, cobalt, and calcium can serve as less effective alternate cation activators. Maximum activity requires a reducing agent, and the enzyme has a broad pH range from 7.0 to 10.5. The molecular weight of the enzyme is

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... tely 400,000, with eight identical subunits. While the enzyme shows no tendency to form multimolecular aggregates as do other liver pyrophosphorylases, the eight subunits are arranged in a stacked tetrameric configuration. Amino acid analysis, peptide mapping, gel electrophoresis, and sedimentation showed the enzyme to be homogeneous with a single NH*-terminal amino acid being serine. The subunits of the rabbit liver enzyme are chemically identical as confirmed by the foregoing analyses. Although UDP-glucose is the most active substrate, the enzyme will catalyze the pyrophosphorylation of a variety of nucleoside diphosphate hexoses. Rates as high as 14% of that for UDP-glucose were obtained using UDP-galactose as a substrate. The ratio of glucose to galactose activity remained constant throughout purification and no evidence was found for a separate enzyme for UDP-galactose. The equilibrium constant in the direction of UDP-glucose formation was 0.16. The apparent Michaelis constants for UDP-glucose, UTP, and glucose l-phosphate were 6.6 x 10p5, 3.8 x 10e5, and 4.6 x lo", respectively, and that for UDP-galacfose was 4.2 x 10m4. UDP-galactose and galactose 1 -phosphate are competitive inhibitors of UDP-glucose and glucose l-phosphate with apparent constants of 2.2 x 1O-4 and 7.3 x 10m3, respectively. Either 8 moles of UDPglucose or UDP-galactose were bound per mole of enzyme, and the bound UDP-galactose could be replaced stoichiometrically by equivalent quantities of UDP-glucose.