Methods for the Isolation of Glycolytic Intermediates by Column Chromatography with Ion Exchange Resins

Bartlett, Grant R.

doi:10.1016/s0021-9258(18)70225-1

Cited by 247 publications

(14 citation statements)

References 30 publications

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“…Only fractions with an Aim/A^q 5 2.4 were pooled, and they were used within 24 h. [l-2H]-D-Glucose 6-phosphate was prepared from [ 1 -2H] -D-glucose by phosphorylation with yeast hexokinase and MgATP (Vanoni etal., 1990). It was purified by adsorption to Dowex 1-X8 (Clform, 200-400 mesh) and elution with 10 mM HC1 (Bartlett, 1959).…”

Section: Methodsmentioning

confidence: 99%

Structure of the NADPH-Binding Motif of Glutathione Reductase: Efficiency Determined by Evolution

Rescigno¹,

Perham²

1994

Biochemistry

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The role of the second glycine residue (Gly-176) of the conserved GXGXXA "fingerprint" motif in the NADPH-binding domain of Escherichia coli glutathione reductase has been studied by means of site-directed mutagenesis. This glycine residue occurs at the N-terminus of the alpha-helix in the beta alpha beta fold that characterizes the dinucleotide-binding domain, in close proximity to the pyrophosphate bridge of the bound coenzyme. Introducing an alanine residue (G176A), the minimum possible change, at this position virtually inactivated the enzyme, as did the introduction of valine, leucine, isoleucine, glutamic acid, histidine, or arginine residues. Only the replacement by serine--a natural substitute for this glycine residue in some forms of mercuric reductase, a related flavoprotein disulfide oxidoreductase--produced a mutant enzyme (G176S) that retained significant catalytic activity. It is conceivable that this is due to a favorable hydrogen bond being formed between the serine hydroxyl and a pyrophosphate oxygen atom. In most of the mutant enzymes, the Km for NADPH was substantially greater than that found for wild-type glutathione reductase, as expected, but this was accompanied by an unexpected decrease in the Km for GSSG. The latter can be explained by the observation that the reduction of the enzyme by NADPH, the first half-reaction of the ping-pong mechanism, had become a rate-limiting step of the overall reaction catalyzed, albeit poorly, by the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Structure of the NADPH-Binding Motif of Glutathione Reductase: Efficiency Determined by Evolution

Rescigno¹,

Perham²

1994

Biochemistry

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“…(2.1 g, 6 mmol) in H20 (30 mL) was treated with 1.0 N aqueous NaOH (12 mL). The solution was allowed to stand for 3 days at 25 °C, adjusted to pH 7.0, diluted to 500 mL, and loaded on a column (25 X 2.5 cm) of Dowex 1-X8 (1.5 mL/min) (Bartlett, 1959). For maximization of recovery, a loading rate of <1.5 mL/min was used.…”

Section: Methodsmentioning

confidence: 99%

“…(1.5 mL/min) (Bartlett, 1959). Stepwise elution with 0.02 N and 0.06 N HC1 washed unwanted material from the column (monitored by A240).…”

Section: Methodsmentioning

confidence: 99%

Stereospecificity of (E)- and (Z)-phosphoenol-.alpha.-ketobutyrate with chicken liver phosphoenolpyruvate carboxykinase and related phosphoenolpyruvate-utilizing enzymes

Duffy¹,

Saz²,

Nowak³

1982

Biochemistry

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“…The solution was warmed to 37 °C, treated with pyruvate kinase (0.443 mg) and hexokinase (0.09 mg), and kept at 37 °C for 34 h. The reaction was monitored for loss of Z-F-P-enolpyruvate by NMR. The solution was heated to 85 °C for 10 min, cooled, filtered, diluted to 60 mL, adjusted to pH 7.9 with 1.0 N KOH, and loaded on a column (8.5 X 0.75 cm) of Dowex 1-X8 (1.5 mL/min) (Bartlett, 1959). A gradient (300 mL) of 0-0.15 N HC1 was used to elute £-F-P-enolpyruvate.…”

Section: Synthesis Of (Ez)-cyclohexy¡ammonium Dihydrogenmentioning

confidence: 99%

Stereoselectivity of interaction of phosphoenolpyruvate analogs with various phosphoenolpyruvate-utilizing enzymes

Duffy¹,

Nowak²

1984

Biochemistry

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The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.

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Methods for the Isolation of Glycolytic Intermediates by Column Chromatography with Ion Exchange Resins

Cited by 247 publications

References 30 publications

Structure of the NADPH-Binding Motif of Glutathione Reductase: Efficiency Determined by Evolution

Structure of the NADPH-Binding Motif of Glutathione Reductase: Efficiency Determined by Evolution

Stereospecificity of (E)- and (Z)-phosphoenol-.alpha.-ketobutyrate with chicken liver phosphoenolpyruvate carboxykinase and related phosphoenolpyruvate-utilizing enzymes

Stereoselectivity of interaction of phosphoenolpyruvate analogs with various phosphoenolpyruvate-utilizing enzymes

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