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