Reaction rate studies of glucose-6-phosphate dehydrogenase activity in sections of rat liver using four tetrazolium salts

Abstract

The reaction rate of glucose-6-phosphate dehydrogenase activity in liver sections from fed and starved rats has been monitored by the continuous measurement at 37 degrees C of the reaction product as it is formed using scanning and integrating microdensitometry. Control media lacked either substrate or both substrate and coenzyme. All reactions were nonlinear; however, subtraction of either of the controls from the test response produced linearity. Differing responses in sections of livers from fed and fasted rats indicate that the appropriate control medium for use in the assay of this dehydrogenase is one lacking both substrate and coenzyme rather than a medium containing coenzyme. The reaction rate was the same with each of the final acceptors. Problems with the diffusion of the formazan of BPST and with the failure to precipitate the formazan of Neotetrazolium make Tetranitro BT and Nitro BT the tetrazolium salts of choice in quantitative dehydrogenase assays.

Posttranslational Regulation of Glucose-6-phosphate Dehydrogenase Activity in Tongue Epithelium

“…The specific reaction is defined as the test minus control reaction (Butcher and Van Noorden 1985), and final reaction product generated in this specific reaction was completely abolished by addition of 10 mM epiandrosterone, a specific inhibitor of G6PD. This means that 6-phosphogluconate dehydrogenase, a following enzyme in the hexose monophosphate shunt that converts the product of G6PD with concomitant production of NADPH, was not involved in production of staining.…”

“…Sections were incubated for 2 min at RT and then rinsed with hot tapwater to remove the incubation medium and to stop the reaction immediately, and were mounted in glycerin-gelatin. Control reactions were performed in the absence of substrate and co-enzyme (Butcher and Van Noorden 1985). To establish the contribution of SH groups in cells to nonspecific formation of final reaction product, 10 mM N -ethylmaleimide (Sigma) was added to control incubation medium .…”

“…Gray values were converted into absorbance values by using a set of neutral density filters (Kodak, Rochester, NY) (Jonker et al 1997). Values of control reactions were subtracted from test values to obtain specific enzyme activity (Butcher and Van Noorden 1985;Geerts et al 1996). A Vanox-T photomicroscope (Olympus; Tokyo, Japan) with a ϫ 2 objective (NA 0.08) was used.…”

Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29

“…Control reactions were incubated in the absence of substrate and coenzyme. 30,31 In situ determination of transketolase activity was performed according to Boren et al 32 Incubation medium contained 18% (w/ v) PVA in 50 mM Tris-HCl buffer, pH 7.6, 5 mM sodium azide, 7.5 mM NAD 1 , 3.7 mM KH 2 PO 4 , 5 mM MgCl 2 , 0.45 mM methoxyphenazine methosulphate, 100 lL substrate mixture (prepared as described above) per mL incubation medium, 0.1 mM TPP and 5 mM NBT. The medium was freshly prepared just before incubation and NBT was added after being dissolved in a heated mixture of DMF and ethanol (1:1) (final dilution of each solvent in the medium was 2% v/v).…”

Differential Activity of NADPH-Producing Dehydrogenases Renders Rodents Unsuitable Models to Study IDH1R132 Mutation Effects in Human Glioblastoma

“…Experiments were performed in duplicate, and the concentrations of the substrates and coenzymes in the incubation media were sufficiently high to ensure maximum velocity (V max ) of the enzyme activities (Stoward and Van Noorden 1991;Van Noorden and Butcher 1991). Control reactions were performed in the absence of substrate (Butcher and Van Noorden 1985).…”