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Glutamic-Alanine and Glutamic-Aspartic Transaminases of Wheat Germ

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Glutamic-Alanine and Glutamic-Aspartic Transaminases of Wheat Germ Vol. 69 METABOLISM OF TETRACHLOROBENZENES 189 3. 1:2:3:5-Tetrachlorobenzene is very slowly REFERENCES metabolized. Only about 5 is oxidized to and % Azouz, W. M., Parke, D. V. & Williams, R. T. (1952). excreted as 2:3:4:6-tetrachlorophenol in 6 days. Biochem. J. 50, 702. Some 14 % is eliminated unchanged in the faeces, Azouz, W. M., Parke, D. V. & Williams, R. T. (1953). 12 % in the breath and 23 % remains in the tissues Biochem. J. 55, 146. after 6 days. There is evidence that some 9 % of the Azouz, W. M., Parke, D. V. & Williams, R. T. (1955). dose is dechlorinated and eliminated in the ex- Biochem. J. 59, 410. pired air as less chlorinated benzenes. Some 5 % of Cameron, G. R., Thomas, J. C., Ashmore, S. A., Buchan, the dose may also be excreted in the urine as di- J. L., Warren, E. H. & Hughes, A. W. M. (1937). and tri-chlorophenols. Injected 1:2:3:5-tetra- J. Path. Bact. 44, 281. chlorobenzene is partly excreted as such in the Conrad-Billroth, H. (1932). Z. phy8. Chem. 19, 76. El Masri, A. M., Smith, J. N. & Williams, R. T. (1958). faeces, probably via the bile. Biochem. J. 68, 587. 4. 1:2:4:5-Tetrachlorobenzene appears to be the Fewster, M. E. & Hall, D. A. (1951). Nature, Lond., 168,78. least readily metabolized of the three isomers. Holleman, A. F. (1920). Ree. Trav. chim. Pays-Bas, 39, 736. Only about 2 % is converted into 2:3:5:6-tetra- Huntress, E. H. (1948). Organic Chlorine Compound3. chlorophenol in 6 days; 48 % of the dose was found London: Chapman and Hall. in the tissues after 6 days, and 16 % was in the Jondorf, W. R., Parke, D. V. & Williams, R. T. (1955). faeces and 2 % in the expired air. Dechlorination Biochem. J. 61, 512. products could account for 15 % of the dose, about Mead, J. A. R., Smith, J. N. & Williams, R. T. (1958). 10 % of the dose appearing in the expired air as less Biochem. J. 68, 61. Parke, D. V. & Williams, R. T. (1955). Biochem. J. 59,415. chlorinated benzenes and 5 % in the urine as di- and Paul, J. (1951). Ph.D. Thesis, University of Glasgow. tri-chlorophenols. Dechlorination of the tetra- Smith, J. N., Spencer, B. & Williams, R. T. (1950). chlorobenzenes is believed to occur in the gut, Biochem. J. 47, 284. probably under the influence of bacteria. Spencer, B. & Williams, R. T. (1950). Biochem. J. 47, 279. Sperber, I. (1948). J. biol. Chem. 172, 441. The work was supported by a grant from the Agri- Stekol, J. A. (1936). J. biol. Chem. 113, 279. cultural Research Council. Tiessens, G. J. (1931). Rec. Trav. chim. Pays-Bas, 50, 112. Glutamic-Alanine and Glutamic-Aspartic Transaminases of Wheat Germ BY D. H. CRUICKSHANK* Botany School, Univer8ity of Cambridge AND F. A. ISHERWOOD Low Temperature Research Station for Research in Biochemistry and Biophysics, University of Cambridge, and Department of Scientific and Industrial Research (Received 18 December 1957) Since the discovery of the transamination reaction In the present investigation a study has been by Braunstein & Kritzmann (1937), transaminase made of some of the properties of two partially systems have been studied inextracts from anumber purified transaminase enzymes from wheat germ. ofplantandanimaltissuesandmicro-organisms.This Wheat germ was chosen as the plant material work has been reviewed by Braunstein (1947) and because Leonard & Burris (1947) had previously Cohen (1951, 1954). Results of many of the early demonstrated that it contained active tranam.inase investigations were conflicting, as crude enzyme systems. The two transaminase systems were as preparations and non-specific quantitative methods follows: were used. Recently much information on the pro- perties of animal and microbial transaninases has (1) L-Glutamic acid+pyruvic acid beenobtainedwithpurifiedenzymepreparations and oc-oxoglutaric acid+ L-alanine specific quantitative methods. There is, however, (catalysed by glutamic-alanine transaminase) littleprecise information on theplanttransaminases. (2) L-Glutamic acid + oxaloacetic acid = * Present address: Division of Food Preservation and c-oxoglutaric acid + L-aspartic acid Transport C.S.I.R.O., Botany School, University of Sydney, Australia. (catalysed by glutamic-aspartic transaminase) 190 D. H. CRUICKSHANK AND F. A. ISHERWOOD I958 These reactions were followed by the specific At specified times samples were removed for analysis. chromatographic methods recently developed by For the estimation of a-keto acids, 0X1 ml. of the enzyme Isherwood & Cruickshank (1954a, b respectively). digest was added to 0.5 ml. of 0011m-2:4-dinitrophenyl- hydrazine in 0-2N-HC1 in ethanol contained in a stoppered centrifuge tube. The cx-keto acids were then estimated by MATERIALS AND METHODS the method of Isherwood & Cruickshank (1954a). For the quantitative determination of the cx-amino acids, a sample Enzyme preparation (0-1 ml.) of the enzyme reaction mixture was added to Commercial wheat germ (kindly supplied by Dr J. Pace of 0.05 ml. of 0-3N-acetic acid. The mixture was centrifuged the Research Association of British Flour Millers, Cereal and samples of the supernatant were used for the estima- Research Station, St Albans) was extracted three times tion of the x-amino acids by the method of Isherwood & with 1-5 vol. of ether and dried at room temperature. Cruickshank (1954b). Removal of the fat caused the original flaky material to Results have been expressed as micromoles of reactant disintegrate into a powder. This defatted powder (15 g.) present in 1 ml. of enzyme digest. The percentage trans- was suspended in 60 ml. of water in a stoppered bottle and amination (% T), used to express transaminase activity, is the mixture agitated gently for 3 hr. by slowly rotating the defined as the percentage of the initial substrate trans- bottle between rollers. The mixture was then centrifuged; aminated during a specified time. This has been determined the milky supernatant was adjusted to pH 5*7 with from the amount of substrate utilized or the amount of 2N-acetic acid and the liquid again centrifuged. The clear, reaction product formed: the results should be identical brown supernatant was brought to pH 7 with 2x-NaOH whichever method is used. and the liquid treated with saturated (NH4)2SO4 (pH 7) at 50. The fraction precipitated between 33 and 66 % satura- RESULTS tion was suspended in a few millilitres ofwater, dialysed for 15 hr. at 5° against 1% (w/v) KCI and finally adjusted to The purified enzyme preparation contained only pH 7-5 or 8-0 with N-NaOH. No oc-keto acids or amino acids glutamic-alanine and glutamic-aspartic trans- could be detected in this preparation by the chromato- aminases. A preliminary examination of the crude graphic methods referred to above. aqueous extract of wheat germ showed that other transaminases were present, for transamination Substrates occurred to a small extent between a-oxoglutaric x-Keto acids. Commercial samples of ac-oxoglutaric acid, acid and valine, leucine, phenylalanine, tyrosine and oxaloacetic acid and pyruvic acid (as sodium pyruvate) tryptophan. These other transaminases were lost were used. Chromatographic examination of their 2:4- during the purification procedure described above. dinitrophenylhydrazones showed that they were free from other keto acids. Immediately before use neutral 0-2M- Measurements in the glutamic-alanine system solutions were prepared (with the addition of dil. NaOH if necessary) and these were diluted to O-1M with 0-2M- Preliminary experiments showed that there was phosphate buffer (KH2PO4-NaOH), pH 7-5 or 8-0. no significant change in any of the reactants cx-Amino acids. Commercial preparations were used. (reaction 1) when incubated singly with the enzyme These were found by paper chromatography to be free preparation and that the value obtained for rate of from other amino acids. For enzyme studies the amino transamination was the same whichever com- acids were prepared as 0*2M neutral solutions and diluted to ponent was determined. Some results are given in 01m immediately before use with 0-2m-phosphate buffer Table 1. (KH2PO4-NaOH), pH 7-5 or 8-0. Pyridoxal phosphate. A sample of the calcium salt of The results indicated that the glutamic-alanine pyridoxal phosphate was kindly provided by Dr E. F. transamination system was not being influenced by Gale, F.R.S., Department of Biochemistry, University of side reactions involving the formation or destruc- Cambridge. Immediately before use, 40Icg. was dissolved tion of any ofthe reactants, and that the estimation in 1 ml. of 0 2M-phosphate buffer, pH 7-5 or 8-0. of any one of the reactants would serve as a measure of the progress of the reaction. Procedure Progress curves for the forward and reverse To small tubes were added 1 vol. (0.2-0.4 ml.) of 0.2m- reactions are shown in Fig. 1. The points on the phosphate buffer (KH2PO4-NaOH), 1 vol. of 01M-ac- curves were obtained by estimating oc-oxoglutaric amino acid solution and 1 vol. oftransaminase preparation, acid and pyruvic acid at each specified time. and the mixture was incubated at 250 for 10 min.; 1 vol. of The initial rates for the forward and reverse 01M-oc-keto acid solution was then added. To test the reactions were almost equal: at 7-5 min. 27-4 %of effect of inhibitors or coenzymes this procedure was the L-glutamic acid had been converted into a- slightly modified. Enzyme solution (2 vol.) was incubated oxoglutaric acid and 26X6 % of the L-alanine into for 15 min. at 250 with 2 vol. of inhibitor or coenzyme and in 2 vol. of phosphate buffer and then 1 vol. of 0-2M-cx-amino pyruvic acid. Equilibrium was reached 30 min., acid and 1 vol.
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