GLUCOSE-6-PHOSPHATE DEHYDROGENASE from Leuconostoc mesenteroides

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Appearance :White amorphous powder, lyophilized
Activity :GradeⅢ 400 U/mg-solid or more (NAD)
Stability :Stable at −20 ℃ for at least one year
Stable at 5 ℃ for at least 6 months (liquid form)
Molecular weight :104,000(two subunits of approx. 55,000) 1,2)
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PREPARATION and SPECIFICATION

AppearanceWhite amorphous powder, lyophilized
ActivityGradeⅢ 400 U/mg-solid or more (NAD)
ContaminantsCreatine 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

StabilityStable at −20 ℃ for at least one year(Fig.1)
Stable at 5 ℃ for at least 6 months (liquid form)(Fig.3)
Molecular weight104,000(two subunits of approx. 55,000) 1,2)
Isoelectric point4.6 2)
Michaelis constants 2)NAD linked1.06×10-4 M (NAD), 5.27×10-5 M (G-6-P)
NADP linked5.69×10-6 M (NADP), 8.1×10-5 M (G-6-P)
StructureNeither cysteine nor cystine residues is present in the enzyme molecule 1) and essential lysine is indicated to be at active site. 3)
InhibitorsAcyl-CoA,4) ATP,4) mental ions etc. (Table 1)
Optimum pH7.8(Fig.4)
Optimum temperature50 ℃(Fig.5)
pH StabilitypH 5.5−7.5 (30 ℃, 17 hr)(Fig.6)
Thermal stabilitybelow 37 ℃ (pH 8.0, 30 min)(Fig.7)
Substrate specificityEither 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

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 7.855 mM (containing 3.3 mM magnesium chloride)
B. NAD solution60 mM (Should be prepared fresh)
C. G-6-P solution0.1 M glucose-6-phosphate (should be prepared fresh)
D. Enzyme diluent5 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 mLTris-HCl buffer, pH 7.8(A)
0.1 mLNAD solution(B)
0.1 mLG・6・P solution(C)
Concentration in assay mixture
Tris-HCl buffer50 mM
G-6-P3.3 mM
NAD2.0 mM
MgCl23.0 mM
BSA33 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 (cm2/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 ℃.]

  • ChemicalConcn.(mM)Residual
    activity(%)
    None100
    Metal salt2.0
    AgNO386
    Ba(OAc)251
    CaCl290
    Cd(OAc)274
    CoCl280
    CuSO466
    FeCl30
    FeSO41
    HgCl284
    MgCl290
    MnCl289
    NiCl289
    Pb(OAc)23
    Zn(OAc)267
    ZnSO453
    KF2.093
    NaF20.098
    NaN320.093
  • ChemicalConcn.(mM)Residual
    activity(%)
    NEM2.091
    PCMB2.096
    MIA2.014
    Iodoacetamide2.00
    EDTA5.094
    (NH4)2SO420.098
    Borate20.095
    o-Phenanthroline2.093
    α,α′-Dipyridyl2.095
    Urea2.093
    Guanidine2.093
    Hydroxylamine2.091
    Na-cholate1.0 %102
    Triton X-1001.0 %100
    Brij 351.0 %4
    SDS0.1 %0
    Tween 200.1 %101
    Span 200.1 %99
    DAC0.1 %0

Ac, CH3CO; NEM, N-Ethylmaleimide; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammoniumchloride.

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (Powder form)

    Fig.2. Stability (Powder form)

    (kept under dry conditions)

  • Fig.3. Stability (Liquid form at 5℃)

    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

    Fig.4. pH-Activity

    30 ℃ in the following buffer solution: pH 5.7-6.8, 15 mM Veronal-CH3COONaHCI;pH 6.8-8.5,50 mM Tris-HCI; pH 8.5-9.5, 50 mM glycine-NaOH

  • Fig.5. Temperature activity

    Fig.5. Temperature activity

    (in 50 mM Tris-HCI buffer, pH 7.8)

  • Fig.6. pH-Stability

    Fig.6. pH-Stability

    30 ℃, 17 hr-treatment with the following buffer solution: pH 5.0-7.8, 30 mM VeronalCH3COONa-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

    Fig.7. Thermal stability

    30 min-treatment with 5.0 mM glycineNaOH buffer, pH 8.0, containing 0.1 % of bovine serum albumin

2. MSDS

3. Tech Data Sheets/Manuals

Size

1 MG, 10 MG, 5 MG

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