Partial purification and some kinetic properties of glucose-6-phosphate dehydrogenase from Phycomyces blakesleeanus

Glucose-6-phosphate dehydrogenase from sporangiophores of Phycomyces blakesleeanus NRRL 1555 (-) was partially purified. The enzyme showed a molecular weight of 85 700 as determined by gel-filtration. NADP+ protected the enzyme from inactivation. Magnesium ions did not affect the enzyme activity. Glucose-6-phosphate dehydrogenase was specific for NADP+ as coenzyme. The reaction rates were hyperbolic functions of substrate and coenzyme concentrations. The Km values for NADP+ and glucose 6-phosphate were 39.8 and 154.4 microM, respectively. The kinetic patterns, with respect to coenzyme and substrate, indicated a sequential mechanism. NADPH was a competitive inhibitor with respect to NADP+ (Ki = 45.5 microM) and a non-competitive inhibitor with respect to glucose 6-phosphate. ATP inhibited the activity of glucose-6-phosphate dehydrogenase. The inhibition was of the linear-mixed type with respect to NADP+, the dissociation constant of the enzyme-ATP complex being 2.6 mM, and the enzyme-NADP+-ATP dissociation constant 12.8 mM.     

A kinetic study of glucose-6-phosphate dehydrogenase.

The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. Kia is 9.0 muM, Ka is 4.8 muM, and Kb is 36 muM. Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E-NADP forms of the enzyme. The KI for the E-ADP complex is 1.9 mM, while the Ki values for the E-ATP and E-NADP-ATP complexes are 7.2 and 4.5 mM, respectively.     

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.     

Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP⁺, with K_m's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K_m of 0.31 mM. The NADP⁺ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K_m and V_max values with increasing NADP⁺, denoting negative cooperativity. The two K_m's for high and low NADP⁺ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP⁺ binding sites.     

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase

Summary This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitaties outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performedin situ.     

Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis.

The three enzymes glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were isolated in high yield from extracts of Zymomonas mobilis. The principal steps in the isolation procedures involved the use of selected dye-ligand adsorbent columns, with affinity elution of two of the three enzymes. Glucokinase and fructokinase are dimeric proteins (2 X 33000 Da and 2 X 28000 Da respectively) and glucose-6-phosphate dehydrogenase is a tetramer (4 X 52000 Da). Some similarities in the structural and kinetic parameters of the two kinases were noted, but they have absolute specificity for their substrates. Fructokinase is strongly inhibited by glucose; otherwise non-substrate sugars had little effect on any of the three enzymes.     

Inhibition studies of glucose-6-phosphate dehydrogenase from tetracycline-producing Streptomyces aureofaciens

NAD-linked activity of glucose-6-phosphate dehydrogenase from both low-producing and high-producing strains of Streptomyces aureofaciens was inhibited by ATP, ADP, AMP and Pi. The inhibition constants indicate that ADP was the most potent inhibitor. The NADP-linked activity remained unaffected even at relatively high concentrations of these inhibitors. All inhibitions of the NAD-linked activity were competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate. The results represent a possible new regulatory mechanism of glucose-6-phosphate dehydrogenase from a streptomycete and emphasize its involvement in the regulation of the biosynthesis of tetracyclines.     

Human glucose-6-phosphate dehydrogenase gene carried on a yeast artificial chromosome encodes active enzyme in monkey cells

Yeast artificial chromosomes (YACs) permit the cloning of large tracts of human DNA. A YAC containing the human glucose-6-phosphate dehydrogenase gene is shown to encode active enzyme, supporting the inference that the YAC conserves the structure of the genomic DNA.     

Rat liver cytoplasmic glucose-6-phosphate dehydrogenase. Steady-state kinetic properties and circular dichroism.

Steady-state kinetic studies including initial velocity, NADPH product inhibition, dead-end inhibition, and combined dead-end and product inhibition measurements with purified rat liver glucose-6-phosphate dehydrogenase indicate a sequential and obligatory addition of substrates in the order of NADP+, glucose-6-P for the catalytic pathway at pH 8.0. Although instability of 6-phosphoglucono-delta-lactone precluded product inhibition experiments which might directly exclude an enzyme-6-phosphoglucono-delta-lactone complex, the absence of an enzyme-glucose-6-P complex suggests that the enzyme-lactone product is unlikely and the release of products is also ordered, with NADPH released last. Consideration of the kinetic constants (Ka = 2.0 muM, Kiq = 13 muM) and cellular concentration of the substrates and products suggests extensive inhibition of the enzyme in vivo and control by the NADPH/NADP+ ratios. Circular dichroism spectra of the enzyme in 20 mM phosphate buffer at pH 7.0 and 25 degrees C indicate 51% helix and 33% pleated sheet structures which is considerably different from results (14% helix) with yeast enzymes.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.     

Isolation and properties of glucose-6-phosphate dehydrogenase from human cultivated cells]

A new procedure for purification of glucose-6-phosphate dehydrogenase resulting in an electrophoretically homogenous preparation made up of 5.10(8) cells (390 mg of protein) is proposed. The enzyme yield is more than 20%. The molecular weights of a subunit and a native enzyme are 55000 and 220000, respectively. The isoelectric point for the protein lies at 4,8. The kinetics of the enzyme thermal inactivation obey the first order equation with the inactivation rate constant of 6.10(-3) min-1.