[42] ADP-glucose pyrophosphorylase from Arthrobacter

Publisher Summary This chapter discusses the synthesis of adenosine diphosphate (ADP)-glucose pyrophosphorylase from Arthrobacter . Enzymatic activity is determined by measuring the synthesis of adenosine triphosphate (ATP)- 32 P from ADP-glucose and P- 32 P i . The ATP is isolated by adsorption onto Norit A and estimated by measuring the radioactivity contained in the Norit. The reagents used, procedure followed, and steps involved in the purification are also described in the chapter. The assay is employed in a number of bacterial extracts, and reasonably good proportionality between enzyme activity and amount of protein is obtained. Potassium fluoride (KF) is added to the reaction mixture to inhibit the inorganic pyrophosphatase in the crude extracts and is not required in the purified enzyme. The pyrophosphorylase contains no detectable amounts of phosphoglucomutase, adenosine triphosphatse (ATPase), aldolase, inorganic pyrophosphatase, and phosphohexoisomerase. Magnesium ++ (Mg ++ ) is necessary for enzyme action. Maximal activity is obtained at 6 × 10 –3 M. The enzyme converts glucose- 14 C-1-P quantitatively into ADP-glucose- 14 C if activator and inorganic pyrophosphatase are added to the reaction mixture.