The Leuconostoc GPDH exhibits dual coenzyme specificity, namely NAD and NADP. When assayed under conditions that are optimal for the particular coenzyme, the ratio of observed catalytic activity is NAD/NADP = 1.8.
Stability/Storage: The Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase is a relatively stable enzyme in solution. The lyophilized and ammonium sulfate preparations are stable for 12 months when stored at 2-8°C.
Chromatographically purified. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities 0.011%, 0.002%, 0.0011% and 0.0011% respectively. A suspension in 3.7M ammonium sulfate.
Glucose-6-Phosphate Dehydrogenase Assay
Method
This dehydrogenase is rather unique in that it possesses dual coenzyme specificity. When assayed under conditions that are optimal for the particular coenzyme, the ratio of observed catalytic activity is NAD/NADP = 1.8. The reaction velocity is determined by measuring the increase in absorbance at 340 nm resulting from the reduction of NAD or NADP. One unit reduces one micromole of pyridine nucleotide per minute at 30°C and pH 7.8 under the specified conditions.
Reagents
0.055 M Tris⋅HCl buffer pH 7.8 containing 0.0033 M magnesium chloride
0.006 M Nicotinamide adenine dinucleotide phosphate, monosodium salt, (NADP), (TPN). Note: NADP may vary in salt form and degree of hydration. Care must be exercised to use an analytical grade and the correct molecular weight. When using NAD, prepare 0.06 M solution.
0.1 M Glucose-6-phosphate. As above, an analytical grade and the correct molecular weight must be used.
5 mM Glycine buffer, pH 8.0
5 mM Glycine buffer, pH 8.0 containing 0.1% bovine serum albumin
Enzyme
Dissolve lyophilized enzyme or dilute enzyme suspension to 1.0 mg/ml in 5 mM glycine buffer, pH 8.0. Care should be taken not to shake the solution as precipitation may result. Dilute further immediately before use in 5 mM glycine buffer, containing 0.1% albumin, to obtain a rate of 0.02 - 0.045 ΔA/min.
Procedure
Adjust spectrophotometer to 340 and 30°C.
Pipette into each cuvette as follows:
0.055 M Tris⋅HCl buffer, pH 7.8 with 0.0033 M MgCl2 2.7 ml
0.006 M NADP (or 0.06 M NAD) 0.1 ml
0.1 M Glucose-6-phosphate 0.1 ml
Incubate in spectrophotometer at 30deg.C for 7 - 8 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml diluted enzyme and record increase in A340/min for 4 to 5 minutes. Calculate A340/minute from the initial linear portion of the curve.
Calculation
B2422008