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Rates of NADPH production via the pentose phosphate cycle were determined in perfused livers from phenobarbital-treated rats by measuring 14CO2 production from [1-14C]glucose infused in the presence and absence of p-nitroanisole (0.2 mM), a substrate for mixed-function oxidation. In the fed state, basal rates of NADPH generation were 34-44 mumol/g/hr. p-Nitroanisole, which was metabolized at rates of 8.9 mumol/g/hr, stimulated pentose cycle-dependent NADPH production by 21-24 mumol/g/hr. Fasting for 24 hr prior to perfusion diminished pentose cycle flux by 80% and largely abolished the stimulation of the pentose cycle by p-nitroanisole. In contrast, rates of p-nitroanisole O-demethylation were only diminished slightly, to 5.7 mumol/g/hr. Fasting decreased hepatic glucose, glucose 6-phosphate, and 6-phosphogluconate contents drastically as expected. Pretreatment of rats with 6-aminonicotinamide, which is metabolized to a potent inhibitor of 6-phosphogluconate dehydrogenase, decreased rates of NADPH generation via the pentose cycle to 6.9 mumol/g/hr but did not alter rates of p-nitroanisole metabolism (8.8 mumol/g/hr). Basal rates of NADPH generation decreased from 38 to 26 mumol/g/hr during infusion of potassium cyanide (2 mM), an inhibitor of mitochondrial energy metabolism. Cyanide also decreased rates of p-nitroanisole O-demethylation by over 60%; however, stimulation of NADPH generation via the pentose cycle by p-nitroanisole was as great in the presence (17-21 mumol/g/hr) as in the absence of cyanide. Since rates of mixed-function oxidation were unaffected after virtually complete inhibition of the pentose cycle with 6-amino-nicotinamide, it is concluded that reducing equivalents for the mixed-function oxidation of p-nitroanisole are not provided by the pentose cycle under these conditions.
1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in vitro was measured and contrasted with the value for the pathway acting in the forward direction. The initial specific rates of the pentose pathway reactions in vitro for the reverse and forward directions are measured. 7. The study which includes carbon balance, time course changes and 14C prediction labelling experiments reports a comprehensive investigation of the mechanism of the pentose pathway acting reversibly.
Studies of the metabolism of four pentoses (D-, and L-arabinose; D-, and L-xylose) in frog muscle at 0 degree C revealed that all are metabolized at extremely slow rates. As a result, the metabolic degradation of these pentoses does not significantly affect their equilibrium distribution in muscle cells at this temperature at least. Of the four stereoisomers, three (L-xylose, D-arabinose, L-arabinose) were found exclusively or almost exclusively in the cell water, demonstrating a rectilinear distribution pattern; the equilibrium distribution coefficients (q-values) obtained from the slopes of these rectilinear distribution curves are 0.256, 0.274, and 0.271 respectively. The fourth pentose, D-xylose, is apparently partially adsorbed. With the aid of the equation for solute distribution according to the association-induction (AI) hypothesis, the data for this sugar can also be fitted with a theoretical curve calculated on the basis of a q-value close to those for the other three pentoses. The close similarity of the q-values of pentoses which are sterically different, but have identical molecular weights, provides further confirmation of the "size rule", a prediction of the polarized multilayer (PM) theory of cell water, according to which, the q-values are as a rule primarily determined by the molecular weights of the solute and to a lesser extent dependent on its stereospecificity.
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 induction of D-xylose, D-ribose, L-arabinose, and D-lyxose isomerases by various sugars was studied to determine the configuration necessary for induction. D-Xylose isomerase was only induced by D-xylose, whereas D-ribose isomerase was induced by D-ribose, L-rhamnose, and L-lyxose. L-arabinose isomerase was induced by L-arabinose, D-galactose, L-arabitol, D-fucose, and dulcitol, whereas D-lyxose isomerase was induced by D-lyxose, D-mannose, D-ribose, dulcitol, and myoinositol. Some compounds such as dulcitol, D-galactose, and D- or L-fucose which do not support growth are still able to serve as inducers for various pentose isomerases.
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1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The K(m) values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and P(i) found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [(14)C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the (14)CO(2) yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.
Activity of the pentose phosphate pathway of glucose metabolism was measured in isolated granular pneumocytes under a variety of metabolic conditions known to alter this pathway in intact lungs. Granular pneumocytes were isolated by trypsinization of rat lungs and maintained in primary culture for 24 hr before use. Cells were incubated for 1 hr at 37 degrees with 5.5 mM glucose specifically labeled as 1-14C, 6-14C, U-14C, or 5-3H for determination of glucose utilization, pentose cycle activity, and partition of CO2 production between mitochondrial and pentose pathways. With control cells, total glucose utilization was 111 +/- 4.8 nmoles X hr-1 X (10(6) cells)-1 (mean +/- S.E., N = 19), and 2.2% was metabolized by the pentose cycle. Pentose cycle CO2 production was 7.3 nmoles X hr-1 X (10(6) cells)-1 representing 34% of total CO2 production. Dinitrophenol (50 microM) stimulated mitochondrial CO2 production 5-fold but had no effect on the pentose cycle activity. Phenazine methosulfate (5 microM) had no effect on mitochondrial activity but stimulated pentose cycle activity 15-fold. Antimycin A (0.4 micrograms/ml) markedly inhibited both pathways. After a 30-min preincubation with paraquat (3 mM), the pentose cycle CO2 production increased to 107 nmoles X hr-1 X (10(6) cells)-1 accounting for 39.6% of glucose utilization and 88.4% of CO2 production. Mitochondrial CO2 production was unchanged with paraquat. These studies demonstrate that the pentose cycle in resting granular pneumocytes accounts for a major fraction of the CO2 production from glucose and that activity of this pathway is regulated by the utilization of cytoplasmic reducing equivalents. Paraquat produces marked stimulation of pentose cycle activity in granular pneumocytes, resulting in maximal utilization of cytoplasmic NADPH.
An investigation of the mechanism and quantitative contribution of the pentose phosphate pathway in the glucose metabolism of Morris Hepatoma 5123C is reported. Morris Hepatoma 5123C has an active non-oxidative segment of pentose pathway as judged by its ability to convert ribose 5-P to hexose 6-P in a standard assay. Based on compliance with qualitative and quantitative criteria, the cells exhibit the L-type pentose pathway reaction sequence rather than the F-type pathway. This compliance included the formation of intermediates characteristic of the L-type pathway, namely arabinose 5-P, octulose mono- and bisphosphates and sedoheptulose 1,7-bisphosphate, during the dissimilation of ribose 5-P to hexose 6-P. The intermediary role of arabinose 5-P was suggested by the incorporation of its carbon into various intermediates and products of the pentose pathway. Intermediary roles for ido octulose mono- and bisphosphates were supported by their participation in the reaction catalyzed by the phosphotransferase enzyme of the L-type pentose pathway. Presence of L-type PP reactions was further affirmed by 14C-prediction labelling experiments using [5-14C]- and [2-14C]glucose as specifically labelled substrates. Using two methods of measurement, the F-type pentose cycle made a negligibly small contribution to glucose metabolism, while the measured value of the L-type pentose pathway accounted for 30% (approx.) of the total glucose metabolism of these cells, a value consistent with the high activity of the enzymes of the L-type pentose pathway in Morris Hepatoma 5123C cells and the very high activity of the non-oxidative segment of the pathway in vitro. The findings validate the proposal that the L-type pentose pathway reactions constitute the non-oxidative segment of the pathway in Morris Hepatoma 5123C cells. Reasons involving pyruvate recycling reactions show why there is low incorporation of 14C-isotope in C-1 of glucose 6-P, when [4,5,6-14C]glucose and [6-14C]glucose are L-type PP test substrates in intact cells.
1. Rates of absorption of two hexose (D-glucose and D-galactose) and two pentose (D-xylose and D-arabinose) sugars were measured by in vivo perfusion, in jejunum, ileum and (distal) caecum, in immature hens conditioned to either a standard (ST) or "high fibre" (ST + 20% grass) diet. 2. Each bird was tested in one intestinal segment with all four (U-14C-labelled, 10 mM) sugars, with either the hexoses preceding the pentoses or vice versa. 3. With all treatments, absorption rates of the hexoses were alike, as were those of the pentoses. Hexose absorption was twice as fast as pentose absorption in jejunum and ileum with both dietary pretreatments, whereas in caecum hexose and pentose rates were similarly high, except when pentose (and its associated fluid transfer) was apparently inhibited by prior hexose absorption with the ST diet. 4. With the ST diet, hexose absorption (per unit length and dry weight) was faster in caecum than in jejunum and ileum, and pentose absorption was also fastest in caecum when all pentose data from testing after hexose were excluded. 5. With the ST/grass diet, hexose absorption was faster in jejunum than in ileum and caecum when expressed per unit length, and pentose absorption was fastest in caecum on a dry weight basis. 6. Hexose absorption was faster in jejunum and slower in caecum with the ST/grass pretreatment than with ST. However, the dietary comparison was not conclusive because it involved birds form (two) different hatches (of similar age and weight) tested at different times.
The colonic cells of the large intestine are one of the most proliferative tissues of the animal body. The pentose pathway has an essential role in cell division and growth being the only pathway forming ribose 5-P necessary for all nucleotide and nucleic acid sunthesis. The pentose pathway may also provide reducing potential as NADPH for biosynthesis and C-3- C-8 glycolyl compounds. The maximum catalytic capacities of the reactions of the non-oxidative pentose pathway for the conversion of ribose 5-P to hexose and triose phosphates by the proximal and distal colon under feeding and starvation regimes are among the highest in the animal body. The qualitative presence of the oxidative pentose pathway was assessed by measurement of the C-1/C-6 ratio value of 1.67-1.82. Enzymes of the F-type and L-type pentose pathways are present in colonocytes and their maximum catalytic activities in colonocyte cytosol are reported. The contribution of the F-type pentose cycle to the total glucose metabolism of colonocytes, measured by the specific yield method, is negligibly low (approximately 1.5%). Colonic epithelial cells use glucose at a high rate (7.1 +/- 0.33 mumol min-1g-1 dry wt) and 79% of the glucose is converted to lactate. Arabinose 5-P has an intermediary role in the formation of keto pentose, sedoheptulose and hexose phosphates from ribose 5-P by colonocyte cytosol. The intermediary and reaction products of [1-13C] ribose 5-P dissimilation by colonocytes is investigated by 13C NMR spectroscopy. The 13C positional isotope distributions show labelling of C-1 and C-3 of hexose 6-phosphates consistent with either the theoretical predictions of the F-type pentose pathway or of the activities of exchange reactions catalysed by transketolase and/or transaldolase. Measurements of exchange reactions showed that the C-1/C-3 labelling of these compounds is mostly, if not wholly, attributable to exchange catalysis by these group transferring enzymes. The results suggest that the F-type PC has little role in the glucose metabolism of colonocytes and pentose phosphate formation may thus occur by a contribution (approx 20% of the total glucose metabolism) by the alternate L-type pathway.
The effect of glutathione on stimulation of pentose phosphate cycle activity during oxidative challenge was evaluated in intact Chinese hamster ovary cells in situ. Glutathione was depleted to varying levels with L-buthionine-[S,R] sulfoximine. The level of stimulation of pentose phosphate cycle activity by exogenous H2O2 (4 mumol/10(7) cells) was dependent on the time of pretreatment with L-buthionine-[S,R] sulfoximine and was proportional to the total glutathione concentration. This was not related to the amount of GSSG, since its level was exceedingly low under conditions where H2O2 stimulated pentose phosphate cycle activity. The amount of GSSG in cells increased after exposure to 10-fold higher concentrations of H2O2 under conditions where there was no stimulation of pentose phosphate cycle activity above the basal level. Paraquat caused stimulation of pentose phosphate cycle activity which was independent of L-buthionine-[S,R] sulfoximine pretreatment and of the glutathione content of cells. The stimulatory effects of both oxidants on pentose phosphate cycle activity appeared to be independent of glutathione reductase activity since they were unaffected in cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea. The inhibitory effect of L-buthionine-[S,R] sulfoximine on stimulation of pentose phosphate cycle activity by H2O2 did not appear to be due to the inhibitor itself, but rather to the overall level of glutathione. Glutathione could have a role in maintaining activity of the pentose phosphate cycle at a level which is appropriate for the severity of the oxidative challenge as well as for the capacity of the cellular antioxidant defenses.
By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.