Uses
Glucose-6-phosphate dehydrogenase, Leuconostoc mesenteroides is used to convert p-glucose-6-phosphate to p-glucono-d-lactone-6-phosphate in presence of NADP+ or NAD+. It is used as an intermediate, chemical and research purpose and also used fo analysis.
Purification Methods
The enzyme is useful for measuring pyridine nucleotides in enzyme recycling. The enzyme from Baker's yeast has been purified by (NH4)2SO4 fractionation, Me2CO precipitation, a second (NH4)2SO4 fractionation, concentration by DEAE-SF chromatography, a third (NH4)2SO4 fractionation and recrystallisation. Crystallisation is induced by addition of its coenzyme NADP, which in its presence causes rapid separation of crystals at (NH4)2SO4 concentration much below that required to precipitate the amorphous enzyme. To recrystallise, the crystals are dissolved in 0.01M NADP (pH 7.3) with (NH4)2SO4 at 0.55 saturation, and the crystals appear within 10 to 60minutes. After standing for 2-3days (at 4o) the (NH4)2SO4 is increased to 0.60 of saturation, and more than 80% of the activity in the original crystals is recovered in the fresh crystals. [Noltmann et al. J Biol Chem 236 1255 1961]. Large amounts can be obtained from rat livers. The livers are extracted with 0.025M phosphate buffer (pH 7.5) and precipitated with 3M (NH4)2SO4 (70% of activity). The precipitate is dissolved in 3volumes of 0.025M phosphate (pH 7.5), dialysed against this buffer + 0.2mM EDTA at 4o for 5hours, then diluted to 1% protein and the nucleic acids are precipitated by addition of 0.4volumes of 1% protamine sulfate. (NH4)2SO4 is added to a concentration of 2M (pH adjusted to 7.0 with NH3), the precipitate is discarded and the supernatant is adjusted to 2.8M (NH4)2SO4, dialysed, and the protein is adjusted to 1% and treated with Ca3(PO4)2 gel. The gel is added in three steps (1.5mL of 0.4% gel/mL per step), and the gel is removed by centrifugation after each addition. The third gel adsorbed 50% of the activity. The gel is eluted with 0.2M phosphate buffer (pH 7.4, 40mL/g of gel; 60% recovery). The extract is precipitated in 3volumes of (NH4)2SO4 (adjusted to 4M) to give enzyme with an activity of 30Wmoles/mg of protein per hour. [Lowry et al. J Biol Chem 236 2746 1961.] The Km values for the yeast enzyme are 20WM for G-6P and 2WM for NADP (Tris pH 8.0, 10-2 M MgCl2, 38o) [Noltmann & Kuby The Enzymes VII 223 1963].
