New reaction sequences for the non-oxidative pentose phosphate pathway.

1. Reactions leading to the formation of 14C-labelled volatile compounds and compounds volatile under acid conditions were investigated in a system actively synthesizing hexose 6-phosphates from [U-14C]ribose 5-phosphate by reactions catalysed by enzymes prepared from acetone-dried powder of rat liver; no reactions involving 14C-labelled volatile compounds were detected. Similarly the fixation of 14C-labelled volatile compounds into hexose 6-phosphate could not be detected. 2. A complete carbon balance was made for the reactants, intermediates and products of the reactions involved in the conversion of ribose 5-phosphate into hexose 6-phosphate by enzymes of rat liver. Five additional intermediates of pentose 5-phosphate metabolism in liver were detected, namely D-manno-heptulose 7-phosphate, D-altro-heptulose 1,7-bisphosphate, D-glycero-D-ido-octulose 1,8-bisphosphate, D-glycero-D-altro-octulose 1,8-bisphosphate and D-arabinose 5-phosphate. 3. D-Arabinose 5-phosphate was found to be utilized by a rat liver enzyme preparation to produce both hexose 6-phosphate and triose phosphate. 4. D-Arabinose 5-phosphate was reversibly converted into other pentose 5-phosphates. Paper chromatographic and enzymic evidence indicated that the conversion involved an enzyme tentatively named arabinose phosphate 2-epimerase, which catalyses the following reaction: D-arabinose 5-P in equilibrium D-ribose-5-P. 5. A variety of rat tissues also utilized D-arabinose 5-phosphate to produce both hexose 6-phosphate and triose phosphate and at a rate comparable with that obtained with D-ribose 5-phosphate. 6. A new reaction sequence for the non-oxidative pentose phosphate pathway in liver is proposed.     

Mechanism and quantitative contribution of the pentose pathway to the glucose metabolism of Morris hepatoma 5123C

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.     

Introduction and metabolism of pentose and hexose phosphates in permeabilized Morris hepatoma 5123TC cells

Metabolism of arabinose 5-P, ribose 5-P and glucose 6-P in permeabilized and resealed Morris hepatoma 5123TC cells was investigated by measuring the contribution of these compounds to nucleic acid biosynthesis. The level of [14C]-arabinose (non-phosphorylated) incorporation into nucleic acids was slight, presumably due to the low activity of the transport system or the absence or low activity of a specific 'kinase' enzyme. The permeabilizing procedure involved the brief treatment of Morris hepatoma 5123TC cells with lysolecithin and resulted in a cell population which was permeable to charged compounds i.e. sugar phosphates and nucleotides, that otherwise could not cross the plasma membrane. The permeabilized (and resealed cells) retained normal cellular morphology and intactness of specific organelles as judged by the maintenance of functional properties. Following permeabilization, these cells resealed when transferred back to normal growth medium, and continued to divide and increase at the same rates as control non-permeabilized cell cultures. The permeabilized cells incorporated deoxyribonucleotides ([methyl -3H]-TTP) into DNA at a linear rate of 0.047 nmol per 10(7) cells min-1, representing 90-100 per cent of the DNA synthesis rate in vivo. The permeabilization technique, when coupled with procedures to establish cell synchrony, permitted the comparative estimate of the contributions of [14C]-labelled arabinose 5-P, ribose 5-P and glucose 6-P to RNA, DNA, amino acids, CO2, lactate and sugar mono- and bisphosphates. The percentage of [14C]-isotope incorporated into total nucleic acids by these three labelled sugar phosphates were 2.3, 4.9 and 6.3 respectively. Possible reasons for the lower incorporation of 14C from arabinose 5-P are given. The results are consistent with the proposal that arabinose 5-P, an intermediate of the L-type pentose pathway activity of 5123TC cells, was incorporated into nucleic acids by its interconversion with ribulose 5-P and ribose 5-P and thus into PRPP. This study represents the first report of sugar phosphate as opposed to free sugar metabolism by tumour cells in culture.     

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.     

Carbon-13-enriched carbohydrates: preparation of triose, tetrose, and pentose phosphates.

Three-, four-, and five-carbon aldononitrile phosphates were prepared, purified, and catalyticlly reduced with palladium--barium sulfate (5%) to the corresponding aldose phosphates in high yields at pH 1.7 +/- 0.1 and atmopsheric pressure. DL-Glyceraldehyde 3-phosphate and the tetrose 4-phosphates were prepared with carbon-13 enrichment at C-1, while the pentose 5-phosphates were prepared with enrichment at C-1 and C-2. Preparations of glycolaldehyde phosphate and d-glyceraldehyde 3-phosphate by lead tetra-acetate oxidation of glycerol phosphate and fructose 6-phosphate, respectively, are described. The proportions of cyclic hemiacetals and linear gem-diol forms of the two- to five-carbon aldose phosphates in aqueous solution are reported. Carbon-13 chemical shifts and carbon--phosphorus and carbon--hydrogen coupling constants for the furanose phosphate ring and linear gem-diol phosphates are reported and discussed. d-[2(-13)C]Ribulose 1,5-bisphosphate and L-[3,4(-13)C]sorbose 1,6-bisphosphate were prepared enzymatically from D-[2(-13)C]ribose 5-phosphate and dl-[1(-13)C]glyceraldehyde 3-phosphate, respectively.     

Efficient synthesis of D-[1'-13C]-ribonucleosides for incorporation into oligo-RNA

Syntheses of the monomer building blocks used for the solid-phase synthesis of specifically 1'-13C-labeled oligoribonucleotides from the D-[1-13C]ribose is presented. Procedure has been used for the selective formation of 2'-O-silylated ribonucleosides. After 5'-O-dimethoxytritylation, the synthesis of D-[1'-13C] ribonucleoside phosphoramidites has been achieved.     

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

¹⁴C-Labelled octulose phosphates were formed during photosynthetic ¹⁴CO₂ fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero- d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though ¹⁴C-labelled d-glycero- d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. ¹⁴CO₂ predominantly labelled positions 5 and 6 of d-glycero- d-ido-octulose 1,8-P₂ consistent with labelling predictions of the modified scheme. The kinetics of ¹⁴CO₂ incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to ¹²CO₂ rapidly displaced the ¹⁴C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO₂.) A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO₂ assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO₂ fixation at rates in excess of 120 μmol h⁻¹ mg⁻¹ Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and d-glycero- d-ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that ¹⁴C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO₂ fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction.     

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 nature of the pentose pathway in liver

[2-14C]Glucose, [3,4-14C]glucose, [5-14C]glucose, [4,5,6-14C]glucose, and [1-14C]ribose were perfused through livers of rats. The rats were fed or fasted and refed. In one experiment the liver perfused was regenerating and in another phenazine methosulfate was in the perfusate. Perfusion was for 30 or 90 min. Glucose from each perfusate and liver glucose-6-P and glycogen were isolated, purified, and degraded. The distributions of 14C in the carbons of the glucoses from the glycogens are similar to the distributions from the glucose 6-phosphates. The distributions of 14C are in accord with metabolism of glucose by the classical pentose pathway and not by the L-type pathway that has been proposed to function in liver.     

The separation of isomeric pentose phosphates from each other and the preparation of d-xylulose 5-phosphate and d-ribulose 5-phosphate by column chromatography

The binding of the 5-phosphates of ribose, arabinose, ribulose, and xylulose to a dihydroxyboryl-substituted cellulose column was investigated under various conditions of pH, salt content, and the presence of ethanol. Ribose 5-phosphate was bound to the column at pH 8.1 or above at a high ionic strength when hydrazine was absent but not when it was present. Xylulose 5-phosphate was bound with hydrazine present while ribulose 5-phosphate only bound strongly when both hydrazine and ethanol were present. The structural basis for this behavior is discussed. Procedures are described for isolating ribulose 5-phosphate from an isomerase equilibrium mixture with ribose 5-phosphate, and for isolating xylulose 5-phosphate from a mixture containing ribulose and ribose 5-phosphates.     

Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5'-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially 'competing' pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of 3H:14C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of D-[1-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate <--> UDP-galactose <--> UDP-glucose <--> glucose 1-phosphate <--> glucose 6-phosphate <--> fructose 6-phosphate, showed a large decrease in 3H:14C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5'-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants.     

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.     

EFFICIENT SYNTHESIS OF D-[1′-13C]-RIBONUCLEOSIDES FOR INCORPORATION INTO OLIGO-RNA

Syntheses of the monomer building blocks used for the solid-phase synthesis of specifically 1′-¹³C-labeled oligoribonucleotides from the D-[1-¹³C]ribose is presented. Procedure has been used for the selective formation of 2′-O-silylated ribonucleosides. After 5′-O-dimethoxytritylation, the synthesis of D-[1′-¹³C] ribonucleoside phosphoramidites has been achieved.     

Pentose phosphate pathway in rat colonic epithelium

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.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

In vivo 13C-NMR studies on the metabolism of the lugworm Arenicola marina

13C-NMR natural-abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C. Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C-NMR spectroscopy using 13C-labelled glucose. During recovery from a hypoxic period [1-13C]glucose was incorporated into glycogen. [1-13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1-13C]glucose led to considerably diminished incorporation of 13C-labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity. 13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C-NMR resonance, the compound was synthesized biochemically. No labelling of glycogen was observed when [3-13C]alanine was injected into the coelomic cavity with similar incubation conditions being used. The 13C of [1-13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C(1H)-NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C-labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized. Lugworms were quite sensitive to the 1H-decoupling field used for obtaining the 13C(1H)-NMR spectra, especially at 11.7 T. Using bi-level composite-pulse decoupling and long relaxation delays, no tissue damage or stress-dependent phosphagen mobilization, as judged by 31P-NMR spectroscopy, was observed.     

Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.     

Oxidation of glucose, ribose, alanine, and glutamate by Leishmania braziliensis panamensis

The metabolism of [1-14C]- and [6-14C]glucose, [1-14C]ribose, [1-14C]- and [U-14C]alanine, and [1-14C]- and [5-14C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of 14CO2 produced from [1-14C]glucose to that from [6-14C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of 14CO2 from [1-14C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of 14CO2 production from [1-14C]glucose was almost linear with time of incubation, whereas that of [6-14C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26 degrees C to 34 degrees C had no appreciable effect on the rates or time courses of oxidation of either [1-14C]- or [6-14C]glucose or of [1-14C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of 14CO2 produced from [1-14C]- to [U-14C]alanine and from [1-14C]- to [5-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of N-fluoroacetyl-d-glucosamine into hyaluronate by rabbit tracheal explants in organ culture

1. Incubation of rabbit tracheal explants with N-[(3)H]acetyl-d-glucosamine and N-acetyl-d-[1-(14)C]glucosamine led to labelling of a number of soluble macromolecular products separable from the medium, after papain digestion, by ion-exchange chromatography. 2. With N-acetyl-d-[1-(14)C]glucosamine in the incubation medium, a neutral glycoprotein, two acidic glycoprotein fractions, hyaluronic acid and a glycosaminoglycan fraction were obtained and all were radioactively labelled. Similar labelling occurred with N-fluoroacetyl-d-[1-(14)C]glucosamine or N-fluoro[(3)H]acetylglucosamine as precursor. 3. Maximal labelling was obtained at 96h after incubation of cultures. N-Fluoroacetyl-glucosamine under these conditions was incorporated into hyaluronate less efficiently than N-acetylglucosamine. 4. With N-fluoroacetyl-d-[1-(14)C]glucosamine as precursor, a hyaluronate component was separated that on enzymic degradation by glycosidases (hyaluronidase, beta-glucuronidase and N-acetyl-beta-hexosaminidase) yielded a (14)C-labelled oligosaccharide fraction together with N-acetyl-d-[1-(14)C]glucosamine and N-fluoroacetyl-d-[1-(14)C]glucosamine, consistent with some exchange of N-acetyl groups having occurred. 5. The results on enzymic degradation of labelled macromolecules by glycosidases suggest that the presence of incorporated N-fluoroacetyl side chains may render the hyaluronate analogue more resistant to hyaluronidase.     

Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes.

High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia.