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The Breakdown of Pyruvate by Cell-Free Extracts of the Rumen Micro-Organism LC

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The Breakdown of Pyruvate by Cell-Free Extracts of the Rumen Micro-Organism LC Vol. 74 METABOLISM OF XANTHURENIC ACID 525 with acid; no direct proof, however, is given that that the hydroxyl group in position 8 is bound to this transformation is brought about by detach- glucuronic acid. ment of a glucuronic residue from the hydroxyl group in position 4 of xanthurenic acid. The fact The authors are grateful to Professor Benassi of the that the R. values of Rothstein & Greenberg's Institute of Pharmaceutical Chemistry of the University of compounds B and D in butanol-acetic acid-water Padova for a kind gift of a sample of 8-methylxanthurenic (4:1:5) differ from those of our compounds indicate acid ethyl ester. that they are not the same substances. The differ- REFERENCES ence might tentatively be ascribed to the fact that xanthurenic acid can be conjugated in the living Baglioni, C., Fasella, P., Turano, C. & Siliprandi, N. (1960). organism in several ways and that differences exist G. Biochim. (in the Press). not only between various species, e.g. the rabbit Block, R. J. & Boiling, D. (1951). The Aminoacid Compo8i- and the rat (Rothstein & Greenberg, 1957), but tion of Protein and Foods, p. 413. Springfield, Mass.: also between different races of the same C. Thomas. species. Consden, R., Gordon, A. H. & Martin, A. J. P. (1944). Biochem. J. 38, 224. SUMMARY Dalglesh, C. E. (1952). Biochem. J. 52, 3. Dalgliesh, C. E. (1955). J. clin. Path. 8, 73. 1. Two major metabolites of xanthurenic acid Daigliesh, C. E. (1956). Biochem. J. 64, 481. were found in the urine of albino rats after intra- Dent, C. E. (1948). Biochem. J. 43, 169. peritoneal administration of xanthurenic acid. Ewing, G. W. & Ste¢k, E. A. (1946). J. Amer. chem. Soc. 2. These metabolites were isolated by chromato- 68, 2181. graphy on Dowex-50 and column electrophoresis on Feigl, F. (1956). Spot Tests in Organic Analysis, 2nd ed., cellulose powder. p. 137. Amsterdam: Elsevier Publishing Co. Kotake, Y., jun. (1957). Clin. Chem. 3, 432. 3. The spectral and chemical properties of the Kotake, Y., jun. & Nogami, K. (1954). J. Biochem., two compounds indicate that one is a derivative of Tokyo, 41, 621. xanthurenic acid in which the hydroxyl group in Rothstein, M. & Greenberg, D. M. (1957). Arch. Biochim. position 8 is bound to sulphate and the carboxyl Biophys. 68, 206. group to glycine; the other compound differs in Smith, J. N. (1953). Biochem. J. 55, 156. The Breakdown of Pyruvate by Cell-Free Extracts of the Rumen Micro-organism LC BY J. L. PEEL Agricultural Research Council Unit for Microbiology, The Univer8ity, Sheffield 10, and Department of Biochemi8try, The Technical Univer8ity of Norway, Trondheim (Received 23 July 1959) The micro-organism LC, isolated from the sheep in LC this intermediate is derived from pyruvate. rumen by Elsden, Volcani, Gilchrist & Lewis Preliminary experiments by S. R. Elsden & C. (1956), ferments lactate and carbohydrates with Warner (unpublished work) indicated that dried the formation of volatile acids of chain length up to preparations catalyse the anaerobic oxidation of hexanoate. Washed cells break down pyruvate, pyruvate to acetate and a more detailed investiga- giving carbon dioxide, acetate and butyrate as the tion of this reaction was thus of importance in main products (Elsden & Lewis, 1953). Supple- relation to fatty acid synthesis in LC. Previous menting the pyruvate with acetate, propionate or studies on the oxidative decarboxylation of pyru- butyrate results in the formation of large amounts vate have revealed certain well-defined differences of butyrate, pentanoate or hexanoate respectively. betweenthesystems fromdifferent micro-organisms. Elsden & Lewis concluded that in LC the higher LC is difficult to place taxonomically (Elsden et al. volatile fatty acids are produced by a mechanism 1956), although it has recently been suggested that similar to that established in Clo8tridium kluyveri it should be placed in the genus Peptostreptococcus by Barker and his colleagues (Barker, 1957 a). (Gutierrez, Davis, Lindahl & Warwick, 1959). It Whereas the latter organism obtains acetyl- bears some biochemical resemblance to clostridia coenzyme A for fatty acid synthesis by the an- but is otherwise quite distinct from all the organisms aerobic oxidation of ethanol, it was presumed that previously used in studies of pyruvate breakdown. 526 J. L. PEEL I960 The LC system was therefore also of interest from obtained by omitting sulphide from the medium used for the standpoint of comparative biochemistry. extraction and for dissolving the precipitate., When This paper describes the action of cell-free ammonium sulphate precipitation at pH 4-5 was required, extracts of LC on pyruvate, and the general pro- 0.1 vol. of M-sodium acetate buffer, pH 4*5, was added to each volume of saturated ammonium sulphate. For pre- perties of the system. Portions of these findings cipitation at alkaline pH, 0.1 vol. of M-dipotassium hydro- have already appeared as brief reports (Peel, 1955, gen phosphate was added to 1 vol. of extract and this 1956, 1958a), in the latest of which it was sug- mixture and the saturated ammonium sulphate were gested that pyruvyl-coenzyme A may be an inter- adjusted to the required pH with N-sodium hydroxide before mediate in pyruvate breakdown. Further work precipitation. has shown that the observations on which this Dialyseswere carried out at 20 and againstO 8 mM-sodium interpretation was based were misleading because sulphide unless otherwise stated. Dialysis sacs of 0 75 in. of the experimental conditions used, and this diameter were placed in a chamber of 500 ml. capacity, matter is dealt with fully in the present paper. which was rocked at 60 cyc./min. The chamber was fitted with an inlet and an outlet and, after it had initially been filled with the dilute sulphide solution, more was passed MATERIALS AND METHODS through from a reservoir at the rate of 1-2 1./hr. Extract of boiled cells. Dried cells of LC were suspended Growth of the organisM. The rumen organism LC, strain 1, in 10 ml. of water/g. of dried cells and heated at 100° for of Elsden et al. (1956), National Collection of Industrial 10 min' Insoluble debris was removed by centrifuging for Bacteria no. 8927, was used throughout the present work. 20 min. at 12 000 g and the resulting extract stored at - 20° The maintenance and large-scale growth of the organism until required. and the preparation of dried cells were as described by Coenzyme A, acetyl-coenzyme A and propionyl-coenzyme A. Walker (1958a), except that the harvested cells were Coenzyme A (CoA), 75% pure, was obtained from Pabst washed in 10 ml. of 0-8 mM-sodium sulphide/g. of wet cell Laboratories, Milwaukee, Wis., U.S.A. Acetyl- and pro- paste. The dried cells were pulverized and stored at - 200; pionyl-CoA were prepared from CoA by treating with the preparations up to 4 years old still retain activity. acid anhydrides and then purifying by paper chromato- Cell-free extracts. Dried cells (2 g. portions) were extrac- graphy, with acetate buffer in ethanol as the solvent ted by suspending in 20 ml. of 0 01 M-potassium phosphate, (Stadtman, 1957). In each case a single thioester zone was pH 6-8, to which was added 0-8 mM-sodium sulphide to obtained on the chromatogram. The adenine content of the prevent oxidation by air. The suspension was incubated in eluate was taken as a measure of the amount of thioester vacuo at 370 for 1 hr., after which insoluble debris was re- present. The value from a corresponding blank strip of moved by centrifuging at 12 000g for 20 min. The resulting paper was less than 2 % of that from the thioester zone. supernatant will be referred to as 'crude extract' and con- Cofactor preparations containing acyl-coenzyme A. Early tained 20-30 mg. of protein/ml., as determined by the experiments on pyruvate oxidation by LC preparations in method of Bucher (1947). For dye-reduction experiments, a dye-reduction system suggested that a new cofactor, crude extracts were treated further at 00 by adding 9 ml. of present in extract of boiled cells, was required and the saturated ammonium sulphate/ml. of extract, centrifuging active material was purified from LC. Examination of the at 12 000 g for 20 min., discarding the supernatant and dis- product showed that the principal active constituents were solving the precipitate in the phosphate-sulphide mixture two thioesters of CoA, most probably the acetyl- and pro- used for extraction. This preparation will be referred to as pionyl-derivatives. Cofactor concentrates from LC were 'ammonium sulphate-precipitated preparation'. subsequently used in several experiments reported here Both types of preparation were stored at 00 when in use, but their preparation will only be outlined because: (a) the or at - 20° for longer periods; they were contained in tubes purified cofactor could be replaced by synthetic acetyl- or and discarded when the depth of liquid fell below 1 cm. propionyl-CoA, or, with a modified experimental system, by Crude extracts were stable under these conditions and re- CoA itself; (b) there were indications that the batch of tained full activity after several months at -20°. The ion-exchange resin used at one stage of the purification was ammonium sulphate-precipitated preparations were less abnormal in its behaviour towards CoA and its derivatives, stable and lost up to 20 % of their activity after 6 hr. at 00. consequently the details of this part of the writer's pro- Duplicate samples stored at - 20° and tested after different cedure are not expected to be generally applicable. periods up to several weeks had the same activity, but if a The starting material was wet cell paste harvested from sample was tested, stored at - 20° and retested, up to 50 % large-scale cultures of LC and was first extracted with of the original activity was lost.
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