Pyridine nucleotides in the thyroid: III. A method for determination of oxidized and reduced diphosphopyridine nucleotides using 6-phospho-[I14C]-gluconate

Abstract

Methods are presented for assay of micromicromolar quantities of oxidized and reduced NAD following synthesis to NADP utilizing excess ATP and NAD kinase (ATP:NAD 2′-phosphotransferase, EC 2.7.1.23). NADP+ is assayed by minor modifications of prior methodology in which the yield of 14CO2 is proportional to NADP+ when excess 6-phospho[I14C]gluconate and excess 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate:NADP+ oxidoreductase, EC 1.1.1.44) are present. The reduced nucleotides are assayed in their oxidized state following reaction with excess α-ketoglutarate, NH4+ and glutamate dehydrogenase (l-glutamate:NAD+ (NADP+) oxidoreductase (deaminating), EC 1.4.1.3). The prior methodology for NADP+ assay has been modified, without any sacrifice to sensitivity, so that incubations can be performed in reduced volumes and at room temperature. These modifications have enabled the assay procedures to be performed in center wells, temporarily sealed in counting vials, thereby eliminating the time consuming step of hyamine transfer. Using these methods equivalent amounts of nucleotides per gram wet weight tissue were obtained with different amount of tissue extract. Recovery of internal standards was virtually complete. Values for a number of mammalian tissues, obtained by utilization of the present methodology, are presented and are similar to those previously reported. The influence of extraction procedure upon nucleotide concentration obtained in tissues is mentioned.