The game of the pentose phosphate cycle

Sugar rearrangement in the pentose phosphate cycle for transformation of six pentoses into five hexoses is analysed by abstraction to a mathematical model consisting of the resolution of a logical mathematical game of optimization. In the model, the problem is to arrive at five boxes containing six balls each, having started with six boxes containing five balls each, where boxes simulate the sugars and balls simulate the carbons in each. This is achieved by means of transferring two or three balls from any box to any other in each step, according to transketolase and transaldolase (or aldolase) mechanisms which account for sugar interconversions in the living cell. A hypothesis of simplicity is imposed in order to arrive at the objective with the least number of steps and with the least number of balls in the intermediary boxes. A symmetrical solution is obtained, demonstrating that this is the simplest solution, which is the procedure carried out by biological systems. The same treatment is applied for sugar rearrangement in the non-oxidative phase of the Calvin cycle in photosynthesis and the analysis of the "L-type" of pentose phosphate cycle is also treated, obtaining similar solutions in both cases, which allow us to make some physiological reflections.     

Regulation of the pentose phosphate cycle in bass (Dicentrarchus labrax L.) liver

The influence in the activities of the glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase produced by the ratio changes NADPH/NADP is studied. The intracellular ratios of 20 and 10 are enough to achieve total inhibition of these two enzymes, respectively. Measurements of a number of metabolic intermediates show that the concentrations of Krebs cycle compounds are higher than those of glycolytic pathway metabolites. From a consideration of these values, the regulation of the pentose phosphate cycle mainly by the intracellular NADPH/NADP ratio, is discussed.     

Reductive pentose phosphate cycle in Nitrosocystis oceanus

Campbell, Ann E. (Woods Hole Oceanographic Institution, Woods Hole, Mass.), Johan A. Hellebust, and Stanley W. Watson. Reductive pentose phosphate cycle in Nitrosocystis oceanus. J. Bacteriol. 91:1178-1185. 1966.-Assays in cell-free extracts of Nitrosocystis oceanus, a marine chemoautotrophic bacterium, have demonstrated the presence of all of the enzymes of the reductive pentose phosphate cycle, with activities high enough to account for the normal growth rate of the cells. Studies on ribulosediphosphate carboxylase activity in these extracts showed that it is inhibited by MgCl(2) (30% at 0.01 m), MnCl(2) (70% at 0.01 m), NaCl and KCl (100% at 0.5 m, 63% at 0.2 m), and by sulfate (35% at 0.01 m); phosphate, glutathione, and ethylenediaminetetraacetic acid had no effect. The bacterial enzyme differs from the spinach enzyme with respect to its affinity for bicarbonate and its pH optimum. Whole cells were incubated with C(14)O(2), and the acid-soluble fraction was analyzed by paper chromatography and autoradiography. Phosphoglyceric acid and the sugar phosphates were the earliest labeled compounds; several amino acids and organic acids were also labeled. It is concluded that N. oceanus incorporates CO(2) primarily via the reductive pentose phosphate cycle.     

Further evidence for the classical pentose phosphate cycle in the liver

Isolated rat hepatocytes were incubated with [3-(14)C]xylitol or d-[3-(14)C]xylulose plus xylitol or glucose at substrate concentrations. The glucose formed was isolated and degraded to give the relative specific radioactivities in each carbon atom. C-4 of glucose had the highest specific radioactivity, followed by C-3, with half to one-fifth that of C-4. Only about 1% of the total radioactivity was in C-1. The data are compared with the predictions of the classical pentose phosphate cycle [Horecker, Gibbs, Klenow & Smyrniotis (1954) J. Biol. Chem.207, 393-403], and the proposed new version of the pentose phosphate cycle in liver [Longenecker & Williams (1980) Biochem. J.188, 847-857], which they denoted as the ;L-type pentose cycle'. The Williams pathway predicts that the specific radioactivity of C-1 of glucose should be half that of C-4 (after correction for approximately equal labelling on C-3 and C-4 of hexose phosphate in the pathway involving fructose 1,6-bisphosphatase). The actual labelling in C-1 is 20-350-fold less than this. When the hepatocytes are incubated with phenazine methosulphate, to stimulate the oxidative branch of the pentose phosphate cycle, the predicted relationship between (C-2/C-3) and (C-1/C-3) ratios of specific radio-activities is nearly exactly in accord with the classical pentose phosphate cycle. Glucose and glucose 6-phosphate were isolated and degraded from an incubation of hepatocytes from starved/re-fed rats with [3-(14)C]xylitol. Although the patterns were of the classical type, there was more randomization of (14)C into C-2 and C-1 in the glucose 6-phosphate isolated at the end of the incubation than in the glucose which was continuously produced.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Activity and metabolic roles of the pentose phosphate cycle in several rat tissues

Activity of the pentose-phosphate pathway in several rat tissues was investigated, developing a new method that gives the activity of each phase (oxidative and non-oxidative) as well as the whole pathway separately. Our results demonstrate that this method is easy to carry out and that it has not the problems of indirect determinations of the previous ones. The activities of the oxidative and non-oxidative phases assayed separately gives us new information on the design of the pathway in the different tissues, from which several conclusions about the physiological role of this pathway can be derived. In all cases the activity of the oxidative phase was much higher than the non-oxidative one, and the global activity of the whole pathway was the same as the activity of the non-oxidative phase. The highest activity was found in lactating mammary gland and adipose tissue. Lung and liver showed to have a moderately high activity. Brain, kidney, skeletal muscle, and intestinal mucosa showed to have also a significant activity although less than other tissues. The switch in the mammary gland from the non-lactating state to the lactating one causes a very high increase of activity of 22 times, remaining the same ratio between the activity of the two phases.     

Operation of the reductive pentose phosphate cycle daring the induction period of photosynthesis in Chlorella

When Chlorella oulgaris ll h cells grown in air containing 4%CO₂ (high-CO₂ cells) were given low concentrations of¹⁴CO₂ (<150ppm), the initial rate of photosynthetic ¹⁴CO₂ fixation wasvery low and linear ¹⁴CO₂ fixation was observed after an inductionperiod which lasted for ca. 45 min. No such induction period was observed when high-CO₂ cells weregiven high concentrations of ¹⁴CO₂ (10,000 ppm) or when IOW-CO₂cells were given either low or high concentrations of ¹⁴CO₂,supporting the observations by Briggs and Whittingham (l). However,irrespective of CO₂ concentrations during growth and of ¹⁴CO₂concentrations during the experiments, most of the ¹⁴C was incorporatedinto phosphate esters during the initial periods of photosynthetic¹⁴CO₂ fixation. These results are in sharp contrast to the reportby Graham and Whittingham (4). ¹ Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received June 30, 1979; )     

Regulation of pentose phosphate pathway dehydrogenases by NADP+/NADPH ratios.

Equilibrium dialysis indicates that rat liver glucose-6-P dehydrogenase binds two molecules of NADP⁺ per subunit with a dissociation constant of 0.6 × 10^?6 M. The NADP⁺ free enzyme will not bind glucose-6-P indicating a compulsory order of substrate binding. Development of an isotopic assay allowed a direct measurement of the effect of physiological alterations in the NADP⁺/NADPH ratio on the activity of glucose-6-P and 6-phosphogluconate dehydrogenases. A combination of enzyme induction and altered NADP⁺/NADPH ratios could produce 30–50 fold changes in the capacity of these enzymes to produce NADPH during alterations in the nutritional state of the animal.     

Regulation of the pentose phosphate cycle. Cofactor that controls the inhibition of glucose-6-phosphate dehydrogenase by NADPH in rat liver.

A cofactor of Mr 10(4), characterized as a polypeptide, was found to co-operate with GSSG to prevent the inhibition of glucose-6-phosphate dehydrogenase by NADPH, in order to ensure the operation of the oxidative phase of the pentose phosphate pathway, in rat liver [Eggleston & Krebs (1974) Biochem. J. 138, 425-435; Rodriguez-Segade, Carrion & Freire (1979) Biochem. Biophys. Res. Commun. 89, 148-154]. This cofactor has now been partially purified by ion-exchange chromatography and molecular gel filtration, and characterized as a protein of Mr 10(5). The lighter cofactor reported previously was apparently the result of proteolytic activity generated during the tissue homogenization. The heavier cofactor was unstable, and its amount increased in livers of rats fed on carbohydrate-rich diet. Since the purified cofactor contained no glutathione reductase activity, the involvement of this enzyme in the deinhibitory mechanism of glucose-6-phosphate dehydrogenase by NADPH should be ruled out.     

Development and intracellular distribution of enzymes of the oxidative pentose phosphate cycle in radish cotyledons

Developmental and compartmentation studies were used to evaluate the relative roles of the oxidative pentose phosphate cycle, the Calvin cycle, and the glycolysis in cotyledons of radish (Raphanus sativus L.).     

The pentose phosphate pathway in rabbit liver. Studies on the metabolic sequence and quantitative role of the pentose phosphate cycle by using a system in situ

1. The reactions of the pentose phosphate cycle were investigated by the intraportal infusion of specifically labelled [(14)C]glucose or [(14)C]ribose into the liver of the anaesthetized rabbit. The sugars were confined in the liver by haemostasis and metabolism was allowed to proceed for periods up to 5min. Metabolism was assessed by measuring the rate of change of the specific radioactivity of CO(2), the carbon atoms of glucose 6-phosphate, fructose 6-phosphate and tissue glucose. 2. The quotient oxidation of [1-(14)C]glucose/oxidation of [6-(14)C]glucose as measured by the incorporation into respiratory CO(2) was greater than 1.0 during most of the time-course and increased to a maximum of 3.1 but was found to decrease markedly upon application of a glucose load. 3. The estimate of the pentose phosphate cycle from C-1/C-2 ratios generally increased during the time-course, whereas the estimate of the pentose phosphate cycle from C-3/C-2 ratios varied depending on whether the ratios were measured in glucose or hexose 6-phosphates. 4. The distribution of (14)C in hexose 6-phosphate after the metabolism of [1-(14)C]ribose showed that 65-95% of the label was in C-1 and was concluded to have been the result of a rapidly acting transketolase exchange reaction. 5. Transaldolase exchange reactions catalysed extensive transfer of (14)C from [2-(14)C]glucose into C-5 of the hexose 6-phosphates during the entire time-course. The high concentration of label in C-4, C-5 and C-6 of the hexose 6-phosphates was not seen in tissue glucose in spite of an unchanging rate of glucose production during the time-course. 6. It is concluded that the reaction sequences catalysed by the pentose phosphate pathway enzymes do not constitute a formal metabolic cycle in intact liver, neither do they allow the definition of a fixed stoicheiometry for the dissimilation of glucose.     

The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle

Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, α-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.     

Evidence for the participation of the reductive pentose phosphate cycle in photoreduction and the oxyhydrogen reaction

The assimilation of ¹⁴C-sodium bicarbonate has been measured in Scenedesmus obliquus as 1) photosynthesis, 2) photoreduction (light dependent incorporation of carbon dioxide by hydrogen adapted cells under conditions where photosynthesis is inoperative), and 3) the oxyhydrogen reaction (dark assimilation of carbon dioxide by hydrogen adapted cells in an atmosphere of hydrogen and 1% oxygen). Degradation of the glucose formed in each of these reactions using the Leuconostoc technique establishes the participation of the reductive pentose phosphate cycle.