Methods

Materials.—The labeled hexoses used in this study were obtained from Nuclear, Chicago and were diluted before use to an activity of approximately 0.05, uc per mg. Carbon-14-labeled glu-cose-6-P was synthesized enzymatically from the corresponding labeled glucose as follows. Yeast hexokinase (Sigma Chemical Co., type V) having an activity of 600,000 Kunitz-McDonald (1946) units/g, in an amount of 2 mg was added to 10 ml of a solution of 5 mM NaHC03, 8 mM ATP, 5 mM MgCl2, and 8mM labeled glucose. The reaction, which was followed manomet-rically by C02 evolution at 38, was complete in about 8 minutes. Adenine nucleotides, inorganic phosphate, and sugar phosphates were precipitated by addition of barium acetate and 4 volumes of ethanol at pH 8.7. The precipitate was recovered by centrifugation and dissolved in a few milliliters of water by shaking with Dowex-50 ion-exchange resin in the H+ form, and the acidic supernatant and washings were adjusted to the phenolphthalein end-point with ammonia. The solution was then passed through a 0.5 X 7.0 cm column of Dowex-1 in the Cl~ form, and, after the column was washed with a 1% glucose solution to dilute out traces of glucose-C14, the hexose phosphates were eluted with 100 mil of 0.05 m NH4C1. The product was obtained by precipitation, as before, with barium acetate and ethanol. After washing with 95% ethanol and drying, a yield of 61% was obtained, consisting of a mixture of roughly 80% glucose-6-P and 20% fructose-6-P (thelatter being dueto hexose isomerase as an impurity in the yeast hexokinase). The material thus obtained was free ofinorganic phosphate, adenine nucleotides, and glucose-C14, and was used without further purification afterconversion to the potassium salt.