[55] l-Glycerol-3-phosphate dehydrogenase from Escherichia coli

This chapter describes the assay method, purification procedure, and properties of L-glycerol-3-phosphate dehydrogenase from Escherichia coli. L-Glycerol-3-phosphate dehydrogenase catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to L-glycerol-3-phosphate (G3P). This reaction is the first step in the biosynthesis of phospholipids from glycolytic intermediates and serves to supply the glycerol backbone. The enzyme is subject to product inhibition by L-glycerol-3-phosphate. The enzyme is assayed routinely by following the disappearance of TPNH spectrophotometrically or the oxidation of G3P by measuring TPNH production fluorometrically. Two purification procedures are presented: the first has been used to study the properties of the enzyme. The second, an abbreviated procedure, is suitable for studies of the enzyme activity in different bacterial strains by removing the interfering TPNH oxidase background. The enzyme was purified approximately 1000-fold; however, a homogeneous preparation was not obtained, as zonal electrophoresis indicated at 3 bands. The purification procedure is found to have copurified triose-phosphate isomerase. A test of the effect of G3P on enzyme activity showed that this compound inhibited DHAP reduction. The enzyme is unstable in dilute salt solution (buffer A), and is inhibited in solutions of high ionic strength. Ammonium sulfate precipitates are routinely resuspended in buffer A and desalted with Sephadex G-25 columns before assay. The enzyme is found to be rapidly inactivated at 52°, a temperature that also inactivates TPNH oxidase.