44] Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides

Abstract

This chapter describes the assay method, purification procedure, and properties of glucose-6-phosphate dehydrogenase from Leuconostoe mesenteroides . The rate of production of reduced coenzyme is determined from the measurements of absorbancy at 340 nm with time in the presence of saturating levels of substrate and coenzyme. Enzyme assays are conducted at 25° in a spectrophotometer with a thermostatted cell compartment. Assay mixtures contain the following components in a final volume of 3.0 ml: 1.0 ml of the Trischloride buffer; 0.2 ml of glucose 6-phosphate; and either 0.2 ml of NADP + or 0.3 ml of NAD + . Reactions are usually initiated by the addition of glucose 6-phosphate or enzyme. The cells are routinely gram-stained and inoculated to a 5% sucrose-AC broth tube prior to the initiation of large-scale growth. The appearance of chains of uniformly gram-positive cocci that produce dextran within 24 hr of growth at 30° in sucrose is considered to be sufficient evidence of a pure culture of Leuconostoc mesenteroides. The purification steps are: disruption of cells, ammonium sulfate fractionation, protamine sulfate treatment, ammonium sulfate fractionation, hydroxyapatite chromatography, and crystallization. The recrystallized enzyme appears to be homogeneous in sedimentation velocity experiments at concentrations of 2–10 mg/ml and in sedimentation equilibrium experiments. The enzyme is quite stable during all stages of purification. The NAD + :NADP + activity ratio remains constant over the course of the purification. The enzyme is inhibited by ATP, but this inhibition is reversed by physiological concentrations of Mg 2+ . Other properties are also discussed.