Pathways of Glucose Dissimilation in Carrot Slices"2 T
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PATHWAYS OF GLUCOSE DISSIMILATION IN CARROT SLICES"2 T. AP REES 3 AND HARRY BEEVERS DEPARTMENT OF BIOLOGICAL SCIENCES, PURDUE UNIVERSITY, LAFAYETTE, INDIANA There is now ample evidence that the pentose dividual carbons of the glucose to other cellular com- phosphate (PP) pathway contributes to the break- ponents. down of glucose in many plant tissues (2, 5). We are ignorant of the precise significance of this se- quence and of its exact relationship to the other MATERIALS AND METHODS main route of glucose catabolism in higher plants, the Embden-Meyerhof-Parnas (EMP) sequence. Carrots of varied origin were bought at the local The likelihood of close interplay between the two store. They were kept at 5° C and used within 4 pathways is recognized since some intermediates and weeks. Very young carrots were not used. Cyl- enzymes are common to both. One result of this is inders of tissue, comprising both phloem and xylem, the possibility that triose phosphate produced from were removed from the tap roots with a No. 4 cork pentose in the PP pathway is converted to pyruvate borer and cut into sections roughly Y4 mm thick. by enzymes normally regarded as belonging to the The discs were cut into distilled water and then rinsed EMP sequence. The extensive metabolism of pentose, twice, dried on paper towels, and divided into random which would result from these reactions, and which samples by careful weighing. These samples were is commonly overlooked in calculating the contribu- used at once without further washing. These prepa- tions of the two pathways, now appears to be of some rations were completed within half an hour. importance in plant metabolism. Viewed in this way In all experiments the tissue was placed in 0.03 M the two sequences differ only in the reactions by potassium phosphate at pH 5.0, 3.0 ml/g fresh weight which they produce triose phosphate. In the EMP of tissue, and maintained at 250 C. Measurements sequence triose phosphates are the only products but of gas exchange were carried out by standard War- in the PP pathway the generation of each mole of burg techniques on 650 mg samples of tissue. Res- triose phosphate is accompanied the reduction of 6 piratory inhibitors were dissolved in 0.03 M phos- moles of triphosphopyridine nucleotide (TPN), the phate and the pH adjusted to 5.0. These solutions release of 3 moles of CO. from C-1 and the regenera- were prepared immediately before use. tion of 2 moles of hexose. Uniformly labeled glucose and glucose-i-C14,-2-C'4 In the work reported in this paper we have ex- and -6-C14 were obtained commercially. Glucose amined the interactions of the two pathways in carrot 3,4-C14 was isolated from 5 day old castor beans tissue. Carrots were chosen because there is already which had been exposed to C1402 in the dark for 5 good evidence that glucose breakdown occurs by both minutes. Under these conditions 90 % of the carbon14 pathways (6, 14, 21). In the first group of experi- incorporated into glucose is confined to carbon atoms ments, attempts were made to alter the balance be- 3 and 4 (19). Pyruvate-1-C'4, -2-C14, -3-C14 were tween the two pathways by using the following selec- obtained from the Radiochemical Center, Amersham, tive agents, arsenite, 2,4-dinitrophenol (DNP), U.K. methylene blue, and iodoacetate. The relative rates In the experiments with labeled substrates 2 ,umoles of release of carbons 1 and 6 of supplied glucose was of the appropriate glucose, or pyruvate, were added the criterion studied in this series of experiments. to 1.3 g of tissue in 4.0 ml of buffer, in Warburg ves- The relative importance of the two pathways in un- sels with a volume of approximately 120 ml. Glu- treated tissue was investigated next by A: measuring cose uptake was estimated by measuring the radio- the progress of the release of C14 as C140, from activity of samples (0.2 ml) which were withdrawn specifically labeled glucoses and pyruvates over long from the external medium at regular intervals. periods and B: determining the contributions of in- The C140, respired by the tissues was absorbed by KOH in the center wells. At intervals the KOH was removed quantitatively and replaced by fresh alkali. The carbonate present in each sample of 'Received for publication April 4, 1960. KOH was precipitated as barium carbonate and 2This work was financed by the Atomic Energy Com- counted. C6/C, ratios were determined as described mission (Contract AT(11-1)-330. by Beevers and Gibbs (7). The measurement of 3Present address: Dewprtment of Botany, University the effects of respiratory inhibitors upon the release of Sydney, Sydney, Australia. of C140, was complicated by their inhibition of glu- 830 AP REES AND BEEVERS-PATHWAYS OF GLUCOSE DISSIMILATION 831 cose uptake. The final concentrations of arsenite and the first, 0.2 ml of the radioactive solution was dried 2,4-dinitrophenol (DNP) chosen almost completely gently in the center of a nickel planchette. This pro- inhibited glucose uptake. In the experiments with cedure was found to be unsatisfactory when a truly these inhibitors the tissue was first supplied with quantitative comparison of the activity of different glucose for 3 to 4 hours in the absence of the in- fractions of a tissue extract was required. Pre- hibitors. The samples were then removed from the sumably, differences in the amounts of solutes present glucose, quickly washed with buffer and placed in in each fraction led to significant variations in the self solutions of the appropriate inhibitor. The concen- absorption of the samples prepared for counting. trations of methylene blue and iodoacetate chosen This method was used only in the estimation of glu- caused only slight inhibition of glucose uptake. cose uptake and in the assessment of activity present Therefore these compounds were added to the tissue in eluates of paper chromatograms. All other meas- with the labeled glucose. Appropriate controls were urements of the activity of C14 were made after it had always run. The values in the tables and graphs are been converted to barium carbonate. Radioactive the averages of duplicate, or in most experiments, compounds and solutions were oxidized to CO2 by triplicate determinations. The results were further wet combustion and the CO, subsequently precipitated confirmed by repeating the individual experiments as barium carbonate. The barium carbonate was with different batches of carrots. counted as an even layer spread over the surface of a porous micro-filter. These measurements were FRACTIONATION OF TISSUE: After removing the corrected for self-absorption of the carbonate. external solution, boiling 80 % ethanol was added to Radioautograms of chromatograms of amino acids the tissue in the Warburg flasks. The tissue was then were prepared. The distribution of label on all other extracted in a Soxhlet apparatus with 200 ml of 80 % chromatograms was determined both by preparation ethanol at 45°C for 48 hours. The ethanol was dis- of radioautograms and by elution of individual com- tilled off and the aqueous solution extracted with a ponents followed by estimation of the activity present few milliliters of hexane to remove pigments. This in each eluate. Compounds were located on chro- aqueous solution was then fractionated into amino matograms by the development of a strip on which acids, organic acids, and sugars by passage through both authentic and the unknown compounds had been columns of ion exchange resins as described by Neal run. and Beevers (15). The fraction of the tissue which was insoluble in 80 % ethanol, was dried to a constant weight in a RESULTS vacuum oven at 500 C. Carefully weighed aliquots were combusted to give an estimate of the total C14 The initial rates of respiration of the carrot slices incorporated into this fraction. The remainder of used in these experiments were similar from day to the fraction, roughly 80 %, was hydrolysed by heating day and averaged 83 microliters O/g fresh weight/ in a sealed tube with water and five times its own hour. Respiratory quotients were frequently deter- weight of Dowex 50 WX10 resin at 1000 C for 50 mined and were always found to be close to 1.0 hours. The contents of the tube were then transfer- Adding glucose at the concentrations used in the red to a -column of the same resin in the acid form main experiments did not lead to any detectable change and the whole thoroughly washed with water. The in the rate of 02 uptake or in the RQ. The absorp- effluent contained the carbohydrates and the acids. tion of micromolar amounts of glucose by slices of The resin was then eluted with 1 N ammonium hy- the kind used here is discussed in a second paper (1). droxide to give the amino acids. In all this work The rate of uptake increased with the development of was solvents were removed and solutions were concen- the induced respiration until the glucose com- trated by boiling under reduced pressure at tempera- pletely absorbed after about eight hours. tures below 400 C. EFFECTS OF INHIBITORS AND OTHER AGENTS ON Each of these main fractions was separated into RATIO: The initial rates of production of its major components by paper chromatography. C6/Cj C14O.2 from glucose-6-C'4 and glucose-i-C14 were Chromatograms were always prepared in duplicate. determined and confirmed a previous observation (7) The amino acids were two separated by directional the ratio between these values was close to 0.50, in- chromatography on Whatman No. 20 paper. Phenol dicating that some glucose was being catabolized by saturated with water was used as the first solvent and the P.P.