Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose

d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [(14)C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [(3)H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.     

Pentose cycle and reducing equivalents in rat mammary-gland slices

1. Slices of mammary gland of lactating rats were incubated with glucose labelled uniformly with (14)C and in positions 1, 2, 3 and 6, and with (3)H in all six positions. Glucose carbon atoms are incorporated into CO(2), fatty acids, lipid glycerol, the glucose and galactose moieties of lactose, lactate, soluble amino acids and proteins. C-3 of glucose appears in fatty acids. The incorporation of (3)H into fatty acids is greatest from [3-(3)H]glucose. (3)H from [5-(3)H]glucose appears, apart from in lactose, nearly all in water. 2. The specific radioactivity of the galactose moiety of lactose from [1-(14)C]- and [6-(14)C]-glucose was less, and that from [2-(14)C]- and [3-(14)C]-glucose more, than that of the glucose moiety. There was no randomization of carbon atoms in the glucose moiety, but it was extensive in galactose. 3. The pentose cycle was calculated from (14)C yields in CO(2) and fatty acids, and from the degradation of galactose from [2-(14)C]glucose. A method for the quantitative determination of the contribution of the pentose cycle, from incorporation into fatty acids from [3-(14)C]glucose, is derived. The rate of the reaction catalysed by hexose 6-phosphate isomerase was calculated from the randomization pattern in galactose. 4. Of the utilized glucose, 10-20% is converted into lactose, 20-30% is metabolized via the pentose cycle and the rest is metabolized via the Embden-Meyerhof pathway. About 10-15% of the triose phosphates and pyruvate is derived via the pentose cycle. 5. The pentose cycle is sufficient to provide 80-100% of the NADPH requirement for fatty acid synthesis. 6. The formation of reducing equivalents in the cytoplasm exceeds that required for reductive biosynthesis. About half of the cytoplasmic reducing equivalents are probably transferred into mitochondria. 7. In the Appendix a concise derivation of the randomization of C-1, C-2 and C-3 as a function of the pentose cycle is described.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

Comparison of Rates of Local Cerebral Glucose Utilization Determined with Deoxy[l-14C]glucose and Deoxy[6-14C]glucose

The activity of the pentose phosphate shunt pathway in brain is thought to be linked to neurotransmitter metabolism, glutathione reduction, and synthetic pathways requiring NADPH. There is currently no method available to assess flux of glucose through the pentose phosphate pathway in localized regions of the brain of conscious animals in vivo. Because metabolites of deoxy[1-14C]glucose are lost from brain when the experimental period of the deoxy[14C]glucose method exceeds 45 min, the possibility was considered that the loss reflected activity of this shunt pathway and that this hexose might be used to assay regional pentose phosphate shunt pathway activity in brain. Decarboxylation of deoxy[1-14C]glucose by brain extracts was detected in vitro, and small quantities of 14C were recovered in the 6-phosphodeoxygluconate fraction when deoxy[14C]glucose metabolites were isolated from freeze-blown brains and separated by HPLC. Local rates of glucose utilization determined with deoxy[1-14C]glucose and deoxy[6-14C]glucose were, however, similar in 20 brain structures at 45, 60, 90, and 120 min after the pulse, indicating that the rate of loss of 14CO2 from deoxy[1-14C]glucose-6-phosphate in normal adult rat brain is too low to permit assay pentose phosphate shunt activity in vivo. Further metabolism of deoxy[1-14]glucose-6-phosphate via this pathway does not interfere during routine use of the deoxyglucose method or explain the progressive decrease in calculated metabolic rate when the experimental period exceeds 45 min.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: II. Evidence for the Participation of Uridine Diphosphoxylose and Free Xylose as Intermediates

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5-(14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5-(14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2-(3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for glucogenesis by germinated pollen tubes. This observation supports the view that free d-xylose is an important intermediate following breakdown of UDP-d-xylose during myo-inositol-linked glucogenesis.     

Metabolism in normal and virus-transformed chick embryo fibroblasts as observed with glucose labeled with 14C and tritium and with tritium-labeled water.

Glucose metabolism in normal and virus-transformed chick embryo fibroblast cells in culture was observed by allowing the cells to metabolize [U-14C]glucose plus glucose labeled with tritium in the C-1, C-3, and C-6 positions. Similarities and differences between normal and transformed cells were observed and measured. Both normal and transformed cells are found to metabolize about 20% of the glucose via the oxidative pentose phosphate cycle, with the rates being about twice as much for transformed cells as for normal cells under the chosen conditions. Nevertheless, the ratio of glucose metabolized via oxidative pentose cycle to the net flow of that metabolized directly to fructose 6-phosphate is about the same in normal and transformed cells. Although the rate of flow of [14C]glucose into the tricarboxylic acid cycle intermediates and amino acids derived from them appears to be the same in normal and transformed cells, the rate of tritium incorporation from H3HO into these intermediates seems to be much higher in normal cells.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

Rates of flux through the pentose cycle in perfused rat liver. A procedure for the calculation of rates of substrate flux from 14CO2 production from [1-14C]glucose

A method for the determination of substrate flux through the pentose cycle was developed employing [1-14C]glucose in experiments with perfused rat livers. The method consists first of a kinetic analysis which differentiates between the production of 14CO2 from [1-14C]glucose via the pentose cycle and via the citrate cycle and, second of a calculation of the specific radioactivity of the hexose monophosphate pool from measured rates of glycolysis and the specific radioactivity of lactate released into the perfusate. The method was validated by experiments comparing the results of tracer infusions with [1-14C]glucose, [6-14C]glucose and [3-14C]pyruvate. In livers from fed rats perfused with 10 mM glucose, the rate of substrate flux through the pentose cycle was around 0.2 mumol X min-1 X g-1; it was about 20% of the substrate flux via glycolysis. The kinetic data were inconsistent with the existence of an L-type pentose cycle in liver.     

Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway

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.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

Quantitative measurement of the L-type pentose phosphate cycle with [2-14C]glucose and [5-14C]glucose in isolated hepatocytes.

1. Investigations of the mechanism of the non-oxidative segment of the pentose phosphate cycle in isolatd hepatocytes by prediction-labelling studies following the metabolism of [2-14C]-, [5-14C]- and [4,5,6-14C]glucose are reported. The 14C distribution patterns in glucose 6-phosphate show that the reactions of the L-type pentose pathway in hepatocytes. 2. Estimates of the quantitative contribution of the L-type pentose cycle are the exclusive form of the pentose cycle to glucose metabolism have been made. The contribution of the L-type pentose cycle to the metabolism of glucose lies between 22 and 30% in isolated hepatocytes. 3. The distribution of 14C in the carbon atoms of glucose 6-phosphate following the metabolism of [4,5,6-14C]- and [2-14C]glucose indicate that gluconeogenesis from triose phosphate and non-oxidative formation of pentose 5-phosphate do not contribute significantly to randomization of 14C in isolated hepatocytes. The transaldolase exchange reaction between fructose 6-phosphate and glyceraldehyde 3-phosphate is very active in these cells.     

The fate of 14C in glucose 6-phosphate synthesized from [1-14C]Ribose 5-phosphate by enzymes of rat liver.

1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1--30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3--17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the 'defined' pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation.     

Estimation of the pentose cycle in the perfused cow''s udder

1. The distributions of (14)C have been compared in the glucose and galactose moieties of lactose obtained from cows' udders perfused with blood containing [1-(14)C]-, [2-(14)C]- and [6-(14)C]-glucose. The (14)C of the glucose moiety was found in the same position as that of the administered glucose, but in the galactose moiety the (14)C from [2-(14)C]glucose was extensively randomized into positions 1 and 3. It is concluded that the glucose moiety arose from free glucose and the galactose moiety from hexose phosphate intermediates and that the latter reflected the randomization occurring through reactions of the pentose cycle. 2. The proportion of the glucose metabolized via the pentose cycle for those cells making lactose was estimated from the distribution of (14)C in the galactose moiety and found to be about 23% in one experiment and 30% in another experiment. 3. The yield and distribution of (14)C were determined in the glycerol of fat from the tissue in experiments with [2-(14)C]- and [6-(14)C]-glucose. There was a greater randomization of (14)C in the glycerol than in C-1, C-2 and C-3 of the galactose moiety of lactose. The ratio of the yield of (14)C in the glycerol from [2-(14)C]glucose to that of [6-(14)C]glucose was very low and from this ratio it was calculated that less than 10% of the glucose was metabolized by the Embden-Meyerhof pathway and approx. 60-70% was converted into lactose. 4. [6-(14)C]Glucose and [6-(3)H]glucose were used to determine whether the (3)H at the C-6 position remained stable during its conversion into glyceride of fat from the tissue. Twenty-seven per cent of the (3)H was labilized during this conversion. Therefore it was not possible to use [2-(14)C]glucose and [6-(3)H]glucose in a single experiment to measure the relative conversion of the C-2 and C-6 positions of glucose to glycerol.     

Incorporation of D-[3-3H, U-14C] glucose into glycerolipid via acyl dihydroxyacetone phosphate untransformed and viral-transformed BHK-21-c13 fibroblasts.

Untransformed BHK-21-c13 fibroblasts as well as 4 polyoma-transformed strains were incubated with D-[U-14C,3-3H]glucose. This substrate generates intracellular labeled glycerol, and also [4-3H]NADPH via the phosphogluconate oxidative pathway. The latter selectively transfers hydrogen to C-2 of glycerol in glycerolipid via the acyl dihydroxyacetone phosphate pathway. After incubation, the distribution of radioactivity and the ratios of 3H/14C at the three positions of recovered glycerol were determined in sn-glycerol 3-phosphate, saponifiable glycerolipids, alkyl ether glycerolipids, and plasmalogens. In each of the cell types examined, 3H in the sn-1 position of glycerol in the recovered ether-containing glycerolipids was negligible, yet this position contained most of the recovered 3H in sn-glycerol 3-phosphate and saponifiable glycerolipids. The 3H/14C ratio in position 2 of glycerol, measured at various incubation times, was from 5- to 200-fold greater in the saponifiable glycerolipids than in free sn-glycerol 3-phosphate. The ratio in position 2 of ether-containing glycerolipids was the same or greater than that in the saponifiable glycerolipids in all of the cell types employed. A similar pattern in the 3H/14C ratio was observed when BHK-21-c13 cells were incubated with D-[U-14C,1-3H]glucose. These observations demonstrate significant participation of the acyl dihydroxyacetone phosphate pathway in glycerolipid synthesis in BHK cells.     

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 utilization of glucose for the synthesis of milk components in the fed and starved lactating goat in vivo.

1. [U-14C]Glucose and [3-3H]glucose were infused into fed and starved lactating goats in order to study glucose metabolism in the mammary gland. 2. Glucose carbon was oxidized and metabolizet to milk lactose, citrate and triacylglycerol in the lactating goat udder. 3. Recycling of glucose carbon in the lactating animal accounted for 10-20% of the total glucose turnover in the whole animal. Recycling of glucose 6-phosphate in the udder accounted for about 25% of the glucose 6-phosphate metabolized. 4. Flux of glucose 6-phosphate through the pentose phosphate pathway was sufficient to account for 34% of the NADPH required for fatty acid synthesis in the gland in the fed animal. 5. Net metabolism of glucose 6-phosphate via the pentose phosphate pathway accounted for 17.8 and 1.2% of the glucose phosphorylated by the mammary gland in the fed and starved animal respectively. Metabolism of glucose 6-phosphate via the pentose phosphate pathway was sufficient to account for all the CO2 produced from glucose in the fed animal, but only 17% of the CO2 produced from glucose in the starved animal.     

Evidence that aldolase and D-arabinose 5-phosphate are components of pentose pathway reactions in liver in vitro

An immunochemical procedure involving the reaction of liver aldolase antibody and rat liver enzyme preparation shows that conversion of ribose 5-P to hexose 6-P by reactions of the non-oxidative pentose pathway fails to occur in the absence of aldolase activity. Radioautography of pentose pathway products formed by liver enzyme catalysis of [U-14C] arabinose 5-P and unlabelled ribose 5-P illustrates the incorporation of 14C into ketopentose, sedoheptulose, fructose and glucose phosphates. There is approximate congruity of the mole specific radioactivity of the pentose and hexose phosphates. These findings are consistent with the proposal that L-pentose pathway reactions constitute the non-oxidative segment of the pathway in liver.     

Use of [2-14C]glucose and [5-14C]glucose for evaluating the mechanism and quantitative significance of the ''liver-cell'' pentose cycle.

1. Expressions are derived for the steady-state measurement of the quantitative contribution of the liver-type pentose phosphate cycle to glucose metabolism by tissues. One method requires the metabolism of [5-14C]glucose followed by the isolation and degradation of glucose 6-phosphate. The second procedure involves the metabolism of [2-14C]glucose and the isolation and degradation of a triose phosphate derivative, usually lactate or glycerol. 2. Measurements of 14C in C-2 and C-5 of glucose 6-phosphate are required and the values of the C-2/C-5 ratios can be used to calculate the quantitative contribution of the L-type pentose cycle in all tissues. 3. The measurement of 14C in C-1, C-2 and C-3 of triose phosphate derivatives can be used to calculate the quantitative contribution of the L-type pentose cycle relative to glycolysis. 4. The effect of transaldolase and transketolase exchange reactions, reactions of gluconeogenesis and non-oxidative formation of pentose 5-phosphate, isotopic equilibration of triose phosphate pools and isotopic equilibration of fructose 6-phosphate and glucose 6-phosphate, which could interfere with a clear interpretation of the data using [2-14C]- and [5-14C]glucose are discussed.     

Anomeric specificity of glucose metabolism in the pentose cycle

The production of 3H2O from alpha- and beta-D-[5-3H]glucose and that of 14CO2 from either alpha- and beta-D-[1-14C] or alpha- and beta-D-[6-14C]glucose were measured in rat pancreatic islets and tumoral insulin-producing cells incubated at 7 degrees C. The ratio in 14CO2 output from D-[1-14C]glucose/D-[6-14C]glucose, the fraction of glucose metabolism occurring through the pentose cycle, and the flow rate through such a cycle were always higher in the presence of beta- than alpha-D-glucose. This indicates that the anomeric specificity of glucose-6-phosphate dehydrogenase is operative in intact islet cells.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.