Monitoring flux through the oxidative pentose phosphate pathway using [1-¹⁴C]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 ¹⁴CO₂ from [1-¹⁴C]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 ¹⁴CO₂ release from either [1-¹⁴C]glucose or [6-¹⁴C]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 [1-¹⁴C]gluconate during metabolism by the cell culture was recovered as ¹⁴CO₂. Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of ¹⁴CO₂ from [1-¹⁴C]gluconate relative to that from [1-¹⁴C]glucose. Thirdly, perturbation of glucose metabolism by glucosamine did not affect ¹⁴CO₂ from [1-¹⁴C]gluconate. Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of ¹⁴CO₂ from [1-¹⁴C]gluconate to a far greater extent than that from [1-¹⁴C]glucose. It is proposed that measurement of ¹⁴CO₂ from [1-¹⁴C]gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.