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The accumulation of 5-keto- d -fructose (5KF) by Gluconobacter cerinus grown on d -fructose in unbuffered medium was shown to be optimal at pH 4.0 after cell growth ceased. During the exponential phase of growth or at neutral pH after the onset of the stationary phase, 5KF production continued but did not accumulate because of its rapid reutilization by reduction to d -fructose. The extent of isotope incorporation into C5 of ribonucleic acid ribose when cells were grown in the presence of specifically labeled d -glucose and d -fructose clearly indicated that (i) the hexose monophosphate oxidative pathway is the predominant metabolic route for carbohydrate assimilation and (ii) extensive randomization of label between C1 and C6 of d -fructose occurred prior to its conversion into pentose. It is suggested that the cyclic oxidation and reduction through the symmetrical 5KF molecule, which accounts for the observed randomization of isotope in d -fructose, provides the cells with an effective mechanism for the regeneration of nicotinamide adenine dinucleotide phosphate during the period of intensive growth.

Direct measurement of 3-O-[3H]methyl-d-glucose uptake by brown fat cells with the use of a rapid filtration procedure was found to provide a suitable index of the d-glucose transport system activity. The rate of net 3-O-methylglucose influx across the fat cell membrane was 10 to 20 times greater than the uptake rate of l-[14C]glucose which enters the cells by simple diffusion. After equilibration the concentration of 3-O-methylglucose in the intracellular water space was the same as that in the medium, indicating that 3-O-methylglucose uptake occurs by facilitated diffusion. Detectable phosphorylation of this d-glucose analogue by brown fat cells under the conditions of these studies did not occur. 3-O-Methylglucose transport was consistently linear for 4 or more min at 23° and was several times greater at 37° and 23° than at 0°. Transport of 3-O-methylglucose was inhibited by d-glucose and by agents known to block d-glucose transport such as phlorizin and cytochalasin B. Both saturable and nonsaturable processes contributed to the initial velocity of 3-O-methylglucose uptake. The saturable component of net influx was estimated by subtraction of uptake rates monitored in the presence of cytochalasin B, which were used to approximate the nonsaturable component, from the initial velocities of 3-O-methylglucose uptake. The corrected values obtained followed simple Michaelis-Menten kinetics with an apparent Kt of about 4 mm. At high concentrations of the hexose, diffusion probably represented the major contribution to uptake since this portion of a Lineweaver-Burk plot of uncorrected values passed through the origin. Insulin markedly stimulated the initial rate of hexose transport, and this effect reflected an increase in apparent Vmax with no detectable difference in apparent Kt. Diamide, H2O2, vitamin K5, and spermine enhanced d-[1-14C]glucose conversion to 14CO2 to a greater extent than d-[6-14C]glucose oxidation, whereas in these experiments insulin increased oxidation of both sugars equally. Vitamin K5 and spermine had little effect on 3-O-methylglucose transport under conditions where insulin, diamide, and H2O2 significantly stimulated this process. The action of diamide and H2O2 to increase 3-O-methylglucose uptake was specific for the d-glucose transport system since, like insulin, they failed to alter l-glucose uptake. The data suggested that vitamin K5 and spermine stimulate fat cell glucose uptake and oxidation primarily by activating intra-cellular metabolic pathways while diamide and H2O2, in addition to an intracellular effect, also activate the d-glucose transport system. These results indicate that glucose uptake in brown fat cells is determined by both the activity of the membrane transport system and the rate of intra-cellular utilization and that alteration of either process can modulate glucose entry rates.

Activities of glucose 6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenases, together with intermediate levels of the glycolytic pathway and the pentose phosphate cycle, were measured throughout a year in the living bark of poplar ( Populus gelrica ). Shoots, immediately after budding (early May), contained very high levels of the three enzyme activities, which fell gradually by early or mid-July to a level, roughly equivalent to budding (May) or growing (July) 2-year-old twigs. In September, the former two dehydrogenase activities of the new shoots and 2-year-old twigs began to rise, while the latter activity started to decrease. The rise of the two dehydrogenase activities continued until late November (or early December). The high level of the two dehydrogenase activities lasted until early in April of the following year and then the decrease in the activities began prior to the onset of budding, reaching a low, basal level in early May. The profile of changes in the two dehydrogenase activities appeared to coincide with the increase and decrease of soluble proteins. Normal concentrations of total hexose phosphates in the glycolytic pathway plus 6-phosphogluconate were found to be 288 to 895 μmoles/kilogram dry weight. During the metabolism transition (September and April), a transient and striking increase of 6-phosphogluconate was observed. In September, 6-phosphogluconate reached a level on the order of 10 −4 m and was 4 times that of fructose 6-phosphate. The increase in 6-phosphogluconate coincided with the increase in the glucose 6-phosphate dehydrogenase activity. Coincidentally, with the change of 6-phosphogluconate level, a large deviation of the in vivo ratio of fructose 6-phosphate to glucose 6-phosphate from the known equilibrium constant was observed, showing the relation of pentose phosphate cycle enzyme activity to the control of glycolysis. The ratio of glucose 6-phosphate to glucose 1-phosphate deviated from that predicted. These ratios fluctuated throughout the year and were affected by the growth phases. The levels of pentose phosphate cycle metabolites, except for 6-phosphogluconate, in the bark were extremely low. The level of inorganic phosphate in the living bark throughout the year was dependent upon the growth phases, suggesting the presence of a regulatory mechanism to maintain inorganic phosphate at a given level as the growth phase changed. Sugar phosphate levels of popular twigs or potato tubers ( Solanum tuberosum ) remained constant after they were stored for 2 weeks at low temperatures, whereas in sweet potato roots ( Ipomoea batatas ), the level rose to about 9-fold of the control, indicating the presence of a strict regulatory system for the synthesis and catabolism of sugar phosphate in the former two.