Partial purification and sugar nucleotide inhibition of UDP-glucose pyrophosphorylase from Lilium longiflorum pollen

Abstract

Uridine diphosphate glucose pyrophosphorylase was purified 34-fold from ungerminated pollen of Lilium longiflorum using ammonium sulfate fractionation, sephadex G-200 gel filtration, and DEAE-cellulose ion exchange column chromatography. Its kinetic properties and inhibition by uridine diphosphate sugars were determined using an isotopic assay which employs retention of the product, UDP-[ 14C] glucose, on DEAE cellulose discs. The Michaelis-Menten constants for the substrates UTP and glucose-1- P were 0.14 m m and 0.46 m m, respectively. Inhibitor studies showed UDP-glucose, UDP-glucuronic acid, UDP-galacturonic acid, UDP-xylose, UDP-galactose, and UDP-mannose to be noncompetitive with the substrate glucose-1- P (only V max decreased), and mixed competitive and noncompetitive ( K m increased, and V max decreased) with the substrate UTP. The apparent inhibition constants ( K i ) were determined to be; UDP-glucose, 0.13 m m; UDP-glucuronic acid, 0.75 m m; UDP-galacturonic acid, 0.93 m m; UDP-xylose, 1.6 m m; UDP-galactose, 4.8 m m; and UDP-mannose, 9.6 m m. In various combinations tested these inhibitors showed simple additive inhibition when present at low individual concentrations (0.4 m m for UDP-galactose, and 0.1 m m for the others) in the presence of low substrate concentrations (UTP at 0.32 m m and glucose-1- P at 0.58 m m). The results of these in vitro studies indicate the possibility that the uridine diphosphate sugars and uridine diphosphate sugar acids, which are the last soluble precursors for cell wall polysaccharide biosynthesis, participate in vivo in a cumulative feedback manner at the UDP-glucose pyrophosphorylase reaction. Such feedback could, in turn, modulate the flow of carbon into pathways leading to cell wall polysaccharides.