Monitoring flux through the oxidative pentose phosphate pathway using [1- 14 C]gluconate

Abstract

The aim of this work was to examine the metabolism of exogenous gluconate by a 4-day-old cell suspension culture of Arabidopsis thaliana (L.) Heynh. Release of (14)CO(2) from [1-(14)C]gluconate was dependent on the concentration in the medium and could be resolved into a substrate-saturable component (apparent K(m) of approximately 0.4 mM) and an unsaturable component. At an external concentration of 0.3 mM, the rate of decarboxylation of applied gluconate was 0.2% of the rate of oxygen consumption by the cells. There was no effect of 0.3 mM gluconate on the rate of oxygen consumption, or on the rate of (14)CO(2) release from either [1-(14)C]glucose or [6-(14)C]glucose by the culture. The following observations argue that gluconate taken up by the cells is metabolised by direct phosphorylation to 6-phosphogluconate and subsequent decarboxylation through 6-phosphogluconate dehydrogenase. First, more than 95% of the label released from [1-(14)C]gluconate during metabolism by the cell culture was recovered as (14)CO(2). Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of (14)CO(2) from [1-(14)C]gluconate relative to that from [1-(14)C]glucose. Thirdly, perturbation of glucose metabolism by glucosamine did not affect (14)CO(2) from [1-(14)C]gluconate. Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of (14)CO(2) from [1-(14)C]gluconate to a far greater extent than that from [1-(14)C]glucose. It is proposed that measurement of (14)CO(2) from [1-(14)C]gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.

Antisense Suppression of the Small Chloroplast Protein CP12 in Tobacco Alters Carbon Partitioning and Severely Restricts Growth

“…To probe the activity of the OPPP, leaf discs were incubated with either [U- 14 C]Glc or [1-14 C] gluconate (Table II). Release of 14 CO 2 from the latter occurs specifically during the decarboxylation of 6-phosphogluconate to ribulose 5-phosphate catalyzed by 6-phosphogluconate dehydrogenase and thus provides a direct measure of flux through the oxidative steps of the pentose phosphate pathway (Garlick et al, 2002). The uptake and metabolism of both substrates was significantly greater in the dark than in the light, and the release of extractable G6PDH activity from leaves sampled in the light or dark: control (black bars), AS2/1 (white bars), and AS6 (gray bars).…”

Glycolytic Enzymes Associate Dynamically with Mitochondria in Response to Respiratory Demand and Support Substrate Channeling

“…This is a well-established response (Averill et al, 1998;Garlick et al, 2002) in which an increased demand for NADPH for nitrite assimilation leads to an increased flux through the OPP pathway via the response of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase to the NADPH/NADP ratio. Titration of KNO 2 concentration against OPP pathway flux in an Arabidopsis cell suspension culture revealed that 5 mM nitrite is sufficient to lead to a significant increase in flux within 3 h ( Figure 3A).…”

“…Flux through the OPP pathway was estimated based on the method of Garlick et al (2002) in which the labeling of CO 2 when cells are incubated with either [1-14 C]gluconate or [6-14 C]glucose is monitored.…”

Oxidative Pentose Phosphate Pathway-Dependent Sugar Sensing as a Mechanism for Regulation of Root Ion Transporters by Photosynthesis

“…6-AN impairs OPPP because it is a potent inhibitor of the phosphogluconate dehydrogenase (Kohler et al, 1970;Garlick et al, 2002), whereas trehalose supply results in an increased accumulation of T6P due to its inhibitory action on T6P phosphatase activity (Schluepmann et al, 2004).…”