E. Racker
1965
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Abstract
Publisher Summary This chapter elaborates the enzymatic determination of D -sedoheptulose-7-phosphate. The method is based on the principle that the determination of sedoheptulose-7-phosphate depends on the four reactions that are described in the chapter. The first reaction is catalyzed by aldolase, second reaction by transaldolase, third reaction by phosphoglucose isomerase, and the fourth reaction by glucose-6-phosphate dehydrogenase. In the presence of excess glyceraldehyde-3-phosphate, which is generated from fructose-1,6-diphosphate, the reaction proceeds until the sedoheptulose-7-phosphate present is completely consumed. Provided that no 6-phosphogluconate is present in the experimental material, 1 μmole sedoheptulose-7-phosphate forms 1 μmole reduced triphosphopyridine nucleotide (TPNH). The chapter emphasizes that glucose-6-phosphate dehydrogenase must not contain 6-phosphogluconic dehydrogenase or TPNH oxidase. The suitability of a preparation is tested as follows: first, the amount of enzyme used in the assay system should catalyze the complete oxidation of 0.05 (μmole of glucose-6-phosphate in less than 5 min; and second, on completion of the reaction in the assay system the optical density at 340 mμ should remain constant for at least 10 min. A continuous decrease in optical density indicates the presence of TPNH oxidase.