CARROT TISSUE AND ETHANOL

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1 CARROT TISSUE AND ETHANOL BY CELIA EOWE AND W. O. JAMES Departments of Botany, Oxford atid Imperial College, London {Received 26 September i%()) (With I figure in the text) SUMMARY The presence of alcohol in an aerated external medium increases the respiration rate of carrot discs. If the alcohol is labelled no lahelling appears in the CO^ emitted or the acids formed in the tissue. Alcohol dehydrogenase is shown to he located almost wholly among the soluble proteins of the cytoplasm. The failure to oxidize alcohol is attrihuted to its spatial separation from the terminal oxidase system located in the mitochondria. INTRODUCTION Carrot discs kept under nitrogen gas have been shown to convert sugars quantitatively to ethanol and carbon dioxide (James and Ritchie, 195). When returned to air the tissues did not oxidize any of the alcohol, which was slowly evaporated into the air stream passing over them (Brierley, 1955). It has been reported that carrot discs immersed in a 5% solution of ethanol at 37 C oxidized it at the rate of 0 mg ethanol per hour per 100 gm tissue (Wetzel, 1933), but unfortunately the methods of investigation were not described. The following experiments were performed to investigate the apparent discrepancy. MATERIALS Carrots were bought in the local market as required. The first batches were bought in Septemher and October and were mature roots; others bought early in the following summer were much younger. The roots were washed, scraped and cylinders of tissue cut out with a 5 mm cork borer. The cylinders were sliced into discs 2 mm thick by means of a cutter with fixed multiple blades. METHODS Gas Exchange. Oxygen consumption and carbon-dioxide output were measured by conventional manometric methods at 30" C. All experiments were done in duphcate and all flasks were sterilized before use. Alcohol was either given in parallel flasks or tipped from the side-arm after an initial period had established the control rate with water alone. Carhon dioxide was estimated hy the direct method. Chromatography. Two-way chromatograms for the identification of organic acids were run as detailed by Elliott (19^)- The lahelling of acids after feeding ethanol-i-"c was examined in one-way paper chromatograms run with alcohol-nha and scanning with the automatic recorder of R. P. Martin. Feeding ivith ethanol-i-"c. Labelled ethanol was obtained from Amersham and

2 Carrot Tissue and Ethanol 289 diluted with unlabelled to give the desired concentration and activities. Four or five gm of carrot discs were incubated in 6 to 10 ml of 5 {, ethanol for to 22 hours at 30" C. The tubes were mechanically shaken and for the last hour of incubation a stream of COj-free oxygen was passed through them and the COj swept over into N NaOH. Recovery of "C. The caustic soda in which the CO., had been trapped was first titrated with N HCl to determine the amount of CO, produced and then converted to barium carbonate which was filtered off using the apparatus of Henriques (Calvin et al., 199). It was aimed to get all samples between 75 and 100 mg to provide a layer of 'infinite thickness' for end-window counting. Combustion was carried out using the apparatus of Beevers, a modification of Calvin's apparatus. In this, CO, from the sample heated with Van Slyke reagent is swept over into Ba(0H)2 by a stream of nitrogen. Owing to the volatility of alcohol it was necessary to freeze the sample in liquid air before adding the Van Slyke oxidation reagent. The carbon dioxide formed was trapped in NaOH or in baryta and the activity determined as before. Samples of tissue were also oxidized for recovery of ''C in the same apparatus. Extraction of alcohol dehydrogenase. Twenty grammes of carrot tissue was ground up in 20 ml of 0.2 M sucrose-phosphate medium at ph 7.2 in a mortar surrounded by ice. The extract was strained off through muslin and a sample of the total homogenate thus obtained was used for determining its alcohol dehydrogenase activity. The remainder of the homogenate was centrifuged at 2500 rev/min for 10 minutes at a temperature of 2"" C to remove cell debris. Mitochondria were obtained from the partially clarified supernatant by centrifuging at 12,500 rev min for 20 minutes and washing once with medium. To 25 ml supernatant after removal of the mitochondria 3 volumes saturated (NH)2SO were added. The protein precipitate was brought down by centrifuging at 10,000 rev/min for 30 minutes. The mitochondrial pellet and the precipitate of soluble protein were resuspended separately in aliquots of the sucrose-phosphate medium for determination of their alcohol dehydrogenase activities. Determination of alcohol dehydrogenase activity. An aliquot of the fraction to be tested was placed in a i cm wide cuvette with 0.5 mg DPN^, a crystal of semicarbazide, 0.05 ml absolute ethanol and M/15 phosphate buffer, ph 7.2, to 3.0 ml. Dehydrogenase activity was measured as increase of optical density at A = 30 mn in a Unicam SP 500 spectrophotometer. RESULTS Oxygen consumption and CO^-oiitput. The addition of ethanol from the side-arm of the respiration flask caused a sudden increase of oxygen uptake during the next 5 or 10 minutes. Thereafter the rate became steady, usually slightly above the rate in water alone. The stabilized rates obtained with varying concentrations of ethanol are shown in Table i. The rates of COa-production are also given. Table i. Gas exchange in v\ per hour of gm carrot tissue in water and dilute alcohol at 30 C. Alcohol tipped from side-arm after i hour Solution Oj-uptake COj-output Water % Ethanol % Ethanol % Ethanol

3 290 CELIA LOWE AND W. O. JAMES In further experiments alcohol was added to parallel flasks at the start and the tissue equilibrated in it. The results (means offive)are given in Table 2. Table 2. Gas exchange in [A per hour by ^gm carrot tissue in water and 0^ alcohol at 30" C Exp. Solution O.j-uptake COj-output a b Water 5% Ethanol Water S% Ethanol Feeding with ethanol-i-"c. A series of seven experiments was performed in which carrot discs were incubated with labelled 5% ethanol as described above. The results of the experiments were all closely similar and are summarized in Table 3. In experiments 6 and 7 the surface of the cut tissue was treated for 30 minutes with 0.3% streptomycin. Table 3. Carrot discs in labelled 5% alcohol tp. Type of tissue Wt. of tissue gm Incubation time hour Activity PC Recovery in count Tissue Acid CO, I z 3 S ,980 10,80 7 o Counts for the extracted acids would not be directly comparable with those obtained from the tissue itself and the emitted CO2; but as no activity was recorded it was not necessar)' to convert the acid extract to carbonate. It is also clear that the slight recoveries of "C in the CO, are negligible in comparison with that taken up and retained by the tissue, which, therefore, has virtually no capacity to oxidize ethanol. Intracellular distribution of alcohol dehydrogenase. The alcohol-dehydrogenase activity of mitochondria and soluble cytoplasmic protein fractions of the cell were determined as described. The results of an experiment using carrots bought from the market in June are shown in Fig. I. A second experiment gave similar results. Very little activity was shown by the mitochondrial fraction. The initial rate shown by the cytoplasmic protein from the same amount of homogenate was at least twenty times faster. DISCUSSION The manonietric results reported above confirm Wetzel's finding that dilute solutions of alcohol increase the gaseous exchange of immersed carrot discs. When the alcohol is labelled no appreciable labelling appears either in the CO, emitted or in the Krebs cycle acids which might be expected to be stages of any oxidation. It must therefore be concluded that the additional CO2 does not come from the alcohol which is not oxidized either fully to CO, or partially to acids. Since all the enzymes necessary for its oxidation are known to be present in carrot

4 Carrot Tissue and Ethanol 291 cells, an explanation for their failure to oxidize it in air is needed. The answer appears to lie quite simply in their spatial distribution. The cytochrome oxidase system operates in carrots and it is known to be firmly anchored to the mitochondrial walls. Alcohol dehydrogenase co-operates with this system itt vitro; but the results given in Fig. i 0 Mitochondrra Time in minutes Fig. I. Tbe alcobol debydrogenase activity of mitocbrondrial and soluble fractions, activity expressed as increase of optical density at 3a mki plotted against time. show that little or none of the enzyme is present in mitochondria. In the absence of air, reduction leading to alcohol formation occurs in the soluble cytoplasm; but re-oxidation in the presence of air fails because the alcohol dehydrogenase and the cytochrome system are held apart. We wish to acknowledge with gratitude a grant from the Nuffield Foundation in support of this work and the kind help of Dr B. C. Loughman of the Department of Agriculture, Oxford. REFERENCES BKIERLEY, ANN F. (1955). D.Pbil. Thesis, Oxford. CALVIN, M., HEIDDBERGER, C, REID, J. C, TOLBERT, B. M. & YANKWICH, P. F. (199). hotopic Carbon. New York. CLAYCOMB, C. K., HUTCHINS, T. T., CATHEY, W. J. & VAN BRIGGER, J. T. (1950). Nucleonics, 7, 1. ELLIOTT, D. C. (195). Detection of glycouic acid in barley shoots. J. Exp. Bot., 5, 353. JAMES, W. O. & RITCHIE, ANN F. (195). The anaerobic respiration of carrot tissue. Proc. Roy. Soc, B, 13, 302. SLATER, W. G. (1955). D.Pbil. Thesis, Oxford. WETZEL, K. (1933). Zur Physiologiederanaeroben Atmung hoherer Pflanzen. Ber. dent. bot. GeselL, 51, 6.

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Blackwell Publishing and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to New Phytologist. A Flavoprotein from Arum Spadix Author(s): W. O. James and Daphne C. Elliott Source: New Phytologist, Vol. 57, No. 2 (Jul., 1958), pp. 230-234 Published by: Blackwell Publishing on behalf of the New Phytologist