GLUCOSE-6-PHOSPHATE DEHYDROGENASE from Leuconostoc mesenteroides

PREPARATION and SPECIFICATION Appearance White amorphous powder, lyophilized Activity GradeⅢ　400 U/mg-solid or more (NAD ＋ ) Contaminants Creatine phosphokinase ≤ 1×10 -3  % Phosphoglucomutase ≤ 1×10 -3  % 6-Phosphogluconate dehydrogenase ≤ 5×10 -3  % Phosphoglucose isomerase ≤ 1×10 -2  % Glutathione reductase ≤ 1×10 -3  % Hexokinase ≤ 1×10 -2  % Myokinase ≤ 1×10 -2  % NADH oxidase ≤ 1×10 -2  % NADPH oxidase ≤ 1×10 -2  % PROPERTIES Stability Stable at −20 ℃ for at least one year (Fig.1) Stable at 5 ℃ for at least 6 months (liquid form) (Fig.3) Molecular weight 104,000(two subunits of approx. 55,000)  1,2) Isoelectric point 4.6  2) Michaelis constants  2) NAD ＋  linked 1.06×10 -4  M (NAD ＋ ), 5.27×10 -5  M (G-6-P) NADP ＋  linked 5.69×10 -6  M (NADP ＋ ), 8.1×10 -5  M (G-6-P) Structure Neither cysteine nor cystine residues is present in the enzyme molecule  1)  and essential lysine is indicated to be at active site.  3) Inhibitors Acyl-CoA, 4)  ATP, 4)  mental ions etc. (Table 1) Optimum pH 7.8 (Fig.4) Optimum temperature 50 ℃ (Fig.5) pH Stability pH 5.5−7.5 (30 ℃, 17 hr) (Fig.6) Thermal stability below 37 ℃ (pH 8.0, 30 min) (Fig.7) Substrate specificity Either NAD ＋  or NADP ＋  serves as coenzyme, the reaction velocity with NAD ＋  being approximately 1.8 times greater than with NADP. +5)  DGlucose-6-phosphate is a preferential substrate for the enzyme, although D-glucose reacts slowly. 6)  Fructose-6-phosphate, fructose1, 6-diphoshate and ribose-5-phosphate are not considered to be substrates. 7) APPLICATIONS This enzyme is useful for enzymatic determation of NAD ＋ (NADP ＋ ) and G-6-P, and the activities of phosphoglucose isomerase, phosphoglucomutase and hexokinase. This enzyme is also used for enzymatic determination of glucose in combination with hexokinase ( HXK-311 ). ASSAY Principle The formation of NADH is measured at 340 nm by spectrophotometry. Unit definition One unit causes the formation of one micromole of NADH per minute under the conditions detailed below. Method Reagents A. Tris-HCl buffer, pH 7.8 55 mM (containing 3.3 mM magnesium chloride) B. NAD ＋  solution 60 mM (Should be prepared fresh) C. G-6-P solution 0.1 M glucose-6-phosphate (should be prepared fresh) D. Enzyme diluent 5 mM Tris-HCl buffer, pH 7.5, containing 0.1 % of bovine serum albumin. Procedure 1.Prepare the following reaction mixture in a cuvette (d ＝ 1.0 cm) and equilibrate at 30 ℃ for about 5 minutes. 2.7 mL Tris-HCl buffer, pH 7.8 (A) 0.1 mL NAD ＋  solution (B) 0.1 mL G・6・P solution (C) Concentration in assay mixture Tris-HCl buffer 50 mM G-6-P 3.3 mM NAD ＋ 2.0 mM MgCl 2 3.0 mM BSA 33 μg/mL 2.Add 0.1 mL of the enzyme solution *  and mix by gentle inversion. 3.Record the increase in optical density at 340 nm against water for 4 to 5 minutes with a spectrophotometer thermostated at 30 ℃ and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test). At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the enzyme diluent is added instead of the enzyme solution. * Dissolve the enzyme preparation in ice-cold enzyme diluent (D) and dilute to 0.05−0.20 U/mL with the same buffer, immediately before the assay. Calculation Activity can be calculated by using the following formula : Volume activity (U/mL) = ΔOD/min (ΔOD test−ΔOD blank)×Vt×df 6.22×1.0×Vs ＝ ΔOD/min×4.82×df Weight activity (U/mg) ＝ (U/mL)×1/C Vt : Total volume (3.0 mL) Vs : Sample volume (0.1 mL) 6.22 : Millimolar extinction coefficient of NADH (cm 2 /micromole) 1.0 : Light path length (cm) df : Dilution facter C : Enzyme concentration in dissolution (c mg/mL) REFERENCES 1)A.Ishaque,M.Mihausen and H.R.Levy; Biochem. Biophys. Res. Comm., 59, 894 (1974). 2)C. Olive, M.E. Geroch and H.R.Levy; J.Biol.Chem., 246, 2043 (1971). 3)M.Milhausen and H.R. Levy; Eur.J.Biochem., 50, 453 (1975). 4)E.L.Coe and L.-H.Hsu; Biochem. Biophys. Res. Comm., 53, 66 (1973). 5)C.Olive and H.R. Levy; Biochem., 6, 730 (1967). 6)R.P.Metzger, S.A. Metzger and R.L. Parsons; Arch Biochem. Biophys., 149, 102 (1972). 7)Methods in Enzymology, Vol, 1, p328 (S.P.Colowick and N.O.Kapalan,eds.), Academic Press, New York (1955). Table 1. Effect of Various Chemicals on Glucose-6-phosphate dehydrogenase ［The enzyme dissolved in 50 mM Tris-HCl buffer,pH 7.5 (5.25 U/mL) was incubated with each chemcal for 1 hr at 30 ℃.］ Chemical Concn.(mM) Residual activity(%) None - 100 Metal salt 2.0 AgNO 3 86 Ba(OAc) 2 51 CaCl 2 90 Cd(OAc) 2 74 CoCl 2 80 CuSO 4 66 FeCl 3 0 FeSO 4 1 HgCl 2 84 MgCl 2 90 MnCl 2 89 NiCl 2 89 Pb(OAc) 2 3 Zn(OAc) 2 67 ZnSO 4 53 KF 2.0 93 NaF 20.0 98 NaN 3 20.0 93 Chemical Concn.(mM) Residual activity(%) NEM 2.0 91 PCMB 2.0 96 MIA 2.0 14 Iodoacetamide 2.0 0 EDTA 5.0 94 (NH 4 ) 2 SO 4 20.0 98 Borate 20.0 95 o-Phenanthroline 2.0 93 α,α′-Dipyridyl 2.0 95 Urea 2.0 93 Guanidine 2.0 93 Hydroxylamine 2.0 91 Na-cholate 1.0 % 102 Triton X-100 1.0 % 100 Brij 35 1.0 % 4 SDS 0.1 % 0 Tween 20 0.1 % 101 Span 20 0.1 % 99 DAC 0.1 % 0 Ac, CH 3 CO; NEM, N-Ethylmaleimide; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammoniumchloride. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. Stability (Powder form) (kept under dry conditions) Fig.3. Stability (Liquid form at 5 ℃) enzyme concentration:5,000 U/mL composition:3.2 M ammonium sulfate,pH 6.0 Fig.4. pH-Activity 30 ℃ in the following buffer solution: pH 5.7-6.8, 15 mM Veronal-CH 3 COONaHCI;pH 6.8-8.5,50 mM Tris-HCI; pH 8.5-9.5, 50 mM glycine-NaOH Fig.5. Temperature activity (in 50 mM Tris-HCI buffer, pH 7.8) Fig.6. pH-Stability 30 ℃, 17 hr-treatment with the following buffer solution: pH 5.0-7.8, 30 mM VeronalCH 3 COONa-HCI;pH 7.5-8.5, 0.1 M Tris-HCI; pH 8.5-9.5,0.1 M glycine-NaOH Fig.7. Thermal stability 30 min-treatment with 5.0 mM glycineNaOH buffer, pH 8.0, containing 0.1 % of bovine serum albumin