cycle intermediates were determined. The degradation of citrate and aspartate C02 FIXA TION IN THE NERVO US SYSTEM*

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1 C02 FIXA TION IN THE NERVO US SYSTEM* BY H. WAELSCH, S.-C. CHENG, L. J. COTEt AND H. NARUSEF NEW YORK PSYCHIATRIC INSTITUTE AND THE COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY Communicated by Henry Lardy, August 18, 1965 Although the effect of CO2 on special structures of the central nervous system (such as the respiratory center and the carotid body) ' and on the peripheral nervous system2 has been recognized by physiologists for many years, the biochemical reactions underlying these effects have not been under close scrutiny, despite the fact that CO2 fixation into the retinal and the occurrence in the brain of enzymes responsible for this process have been demonstrated in the past.4 1 Several years ago, while studying the fate of ammonia administered intravenously to paralyzed cats, we found that the glutamic acid-glutamine system in the brain represented the main removal mechanism for ammonia and that the suspected depletion of intermediates of the citric acid cycle was partly compensated for by increased CO2 fixation.5' 6 In an effort to evaluate further the significance of CO2 fixation in nervous tissue, we have investigated the effect of ionic changes and the addition of drugs to the incubation medium on CO2 fixation in the nonmyelinated nerve of the lobster, as well as the effect of electric stimulation on CO2 fixation in the myelinated sciatic nerve of the bullfrog. In later experiments the lobster and rabbit nerves were used to study the distribution of C14 in citrate and aspartate from CO2 fixation. In these investigations the specific activities of aspartic acid, glutamic acid, and some of the Krebs cycle intermediates were determined. The degradation of citrate and aspartate was done to establish the quantitative aspects of CO2 fixation in the tricarboxylic acid cycle under various experimental conditions. In this report we wish to describe these results and to discuss a possible mechanism by which CO2 may control the rate of the citric acid cycle as well as the concentration of citrate in nervous tissue and secondarily that of acetyl CoA and acetylcholine. Methods.-Paired nerves from the walking legs of lobsters, and sciatic nerves from bullfrogs and rabbits were used in these experiments. The nerves were incubated in their appropriate Ringer's solution containing C14-bicarbonate and the supernatant of the TCA extract was analyzed.7a 7f, 8 The method for the separation of glutamic, aspartic, malic, and succinic acids on Dowex-1-acetate columns was described in a previous paper" and recently extended in order to separate citrate, fumarate, oxalacetate.7d Determination of acids: The determinations of aspartate and malate have already been described.8 The concentration of glutamic acid was determined enzymatically by measuring the reduction of DPN at 340 mjs in the presence of glutamic acid dehydrogenase. This method was accurate in the range of 2-25 mnumoles.7a The microdetermination of citrate was achieved by converting the acid to a-ketoglutarate with the aid of aconitase, isocitrate dehydrogenase, and TPN. The TPNH formed was measured fluorophotometrically. Since the aconitase preparation obtained from the method of Anfinseng contained citrate, ammonia, and other enzymes which interfered with the fluorescence of TPNH, further purification of the aconitase preparation was carried out on a Sephadex B-25 column.id This method was accurate down to 1 m/amole of citrate. The radioactivities of the various samples were determined by standard procedures. Degradation of citrate, aspartate, and glutamate: Citrate was degraded to a-ketoglutarate with 1249

2 1250 BIOCHEMISTRY: WAELSCH ET AL. PROC. N. A. S. the aid of purified aconitase and isocitrate dehydrogenase and subsequently converted to glutamfe in the presence of glutamic acid dehydrogenase, ammonia, and DPNH. When the method was applied to a commercial sample of citrate-1-5 C14, 95% of the radioactivity was found in C-1 and 0-5. This method was suitable for the degradation of micro amounts of citrate.7e Aspartic and glutamic acids were degraded by aspartic acid decarboxylase (Cl. welchii NCTC 6784) and glutamic acid decarboxylase (E. coli ACTT11146), respectively.7e Results and Discussion.-We have found a significant amount of CO2 fixation in the amino acids and Krebs cycle intermediates of the nerves of the lobster, bullfrog, and rabbit. Furthermore, the amount of CO2 fixation can be altered by varying the ionic composition of the incubation medium, by drugs, and by electric stimulation. The results of the nonmyelinated nerve of the lobster are summarized in Table 1. It can be seen that ouabain, or the replacement of Na+ in the incubation medium by choline, Tris, or Li+, suppressed CO2 fixation, whereas increased labeling occurred in a medium which had no added calcium. Acetylcholine, veratrine, and acetylcholine with d-tubocurarine all significantly increased the amount of CO2 fixed in most of the organic acids studied. The electric stimulation of 8 pairs of frog nerves produced an increase of about 24 per cent in the labeling of glutamic acid, without a change in its concentration.ra The detailed analysis of the changes in CO2 fixation produced by environmental conditions cannot be explained simply by a change in the rate of the tricarboxylic acid cycle; however, if only the direction of the changes is considered, the results are consistent as a group. They indicate a possible linkage between ionic movements across the membranes, CO2 fixation, and the operation of the tricarboxylic acid cycle. These findings are consistent with those of Whittam'0 and suggest that the sodium-potassium transport in the membranes is closely linked to the energy metabolism of the nerve cell. The relationship between the metabolism and the ionic movements of the cell offers an explanation for the frequently observed degradative effect of stimulation on the nerve cell. Furthermore, it suggests that the energy metabolism of the cell is equally dependent on the proper ionic distribution in its subcellular organelles. In order to estimate the contribution of C14 in citrate from CO2 fixation at oxaloacetate or oxalosuccinate levels, the acid was isolated from the nerves suspended in bicarbonate-containing medium, and the specific activity, as well as the distribution TABLE 1 CHANGES IN C02 FIXATION IN THE LOBSTER NERVE* ASSOCIATED WITH ALTERED ENVIRONMENTAL CONDITIONS _- % Deviation from Control - No. Condition expts. Totalt Glutamatet Aspartatel Succinatet Malate4 High K (110 mm) Na + choline Na + tris Na + Li Ca Ouabain (0.4 mm) Veratrine (0.04%) Acetylcholine (1 and 5 mm) d-tubocurarine ( mm) Acetylcholine (5 mm) with d-tubocurarine ( mm) * Paired nerves from the same animal were used, one nerve serving as control. t Cpm/mg protein. I Cpm/pmole.

VOL. 54, 1965 BIOCHEMISTRY: WAELSCH ET AL. 1251 TABLE 2 DEGRADATION OF C14-CITRATE OBTAINED FROM LOBSTER AND RABBIT NERVES.

3 VOL. 54, 1965 BIOCHEMISTRY: WAELSCH ET AL TABLE 2 DEGRADATION OF C14-CITRATE OBTAINED FROM LOBSTER AND RABBIT NERVES.- Specific Activity (cpm/m)- _ Citra-tet Animal Glutamate* (C-1) (C-1 + C-6) Ratiot of C-6 to C-1 Lobster :1 It :1 Rabbit (intact) :1 " " :1 Rabbit (damaged) :1 it It :1 * Labeled in the C-1 position only. t Labeled in the C-1 and C-6 positions. $ Ratio of C-6 to C-1 was obtained by (C-6 + C-1 - C-1)/C-1. Incubation time for lobster nerve 0.5 hr; for rabbit nerve 2 hr. of C14 in the acid, were analyzed. The specific activity of citrate was highest of all the acids measured, and therefore a considerable amount of CO2 fixation occurred at the oxalosuccinate level. In lobster nerve the ratios of the average of the specific activities of citrate, malate, aspartate, and glutamate were 50:15:3:1, respectively,?e and in the rabbit nerve the ratios were 6:2: 1: i7f The ratio of the specific activities of citrate to malate indicates that malic acid is not the major site of CO2 fixation. In 2-hr experiments the specific activity of citrate obtained from the rabbit nerve was nearly '/3 of the specific activity of the C14-bicarbonate in the medium.7 The distribution of C14 in citric acid is shown in Table 2. The radioactivity in glutamic acid is solely in C-1, while in citric acid it is in C-1 and C-6. Therefore, the ratio of the specific activity of citrate to glutamate is C-6 + C-1 to C-1. The significance of CO2 fixation in the nervous system lies, as will be discussed further, in its relation to the synthesis of citrate through the carboxylation of a-ketoglutarate. In order to estimate the dilution effect by the carboxylic acid shuttle to the radioactivity in the C-6 position, aspartic acid was degraded and checked for counts (Table 3). The results show that the dicarboxylic acid shuttle in the nerve of the rabbit is almost complete and consistent with the findings reported in mammalian brain.5 However, in the lobster nerve the ratio of the specific activities of C-4 to C-1 of aspartic acid is 10:1 (Table 3) which suggests the absence of an effective dicarboxylic acid shuttle and a low activity of the citrate cleavage enzyme. The radioactivity in C-6 of citrate results from the carboxylation of a-ketoglutarate (Fig. 1). The importance of the carboxylation of a-ketoglutarate was confirmed in the rabbit nerve as well as in the lobster nerve. TABLE 3 But the ratio of the specific activity of DECARBOXYLATION OF C14-ASPARTATE OBTAINED FROM LOBSTER AND RABBIT NERVES Radioactivity in aspartate Radioactivity in a-alanine Animal (cpm) (cpm) Lobster It Rabbit (intact) " " Rabbit (damaged) it it Rabbit and lobster nerves were incubated in C14 bicarbonate Ringer's solution of similar specific activity for 0.5 and 2 hr, respectively. The content of aspartic acid in lobster nerve is approximately 10 times that in rabbit nerve.

4 1252 BIOCHEMISTRY: WAELSCH ET AL. PROC. N. A. S. 8.A. D.C.A.S. O.A.A.+ 4C-C5 C.C.E. 'A +a.2 CIC '- (3) 2 C-C 1 (6) CH 3 C 6 4C-C5 3 C C-C (2) 3 C-C 4 (1) 2 C-C 1 2 CHC 1 ~~~C.A ~~ir 2 C-C 1 K.G. 3 C-C 4 A.A. FIG. 1.-Carbon skeletons of citrate and related acids. A, C14 derived from C4-C02, fixed at oxaloacetate; X, C14 derived from CU4CO2, fixed at oxalosuccinate; +, the number of carbon skeletons of oxaloacetate is started from that of aspartate, and the number in parentheses corresponds to that of citrate; K.G., a-ketoglutarate; C.A., citrate; A.A., aspartate; S.A., succinate; C.C.E., citrate cleavage enzyme; C.E., condensing enzyme; O.A.A., oxaloacetate; D.C.A.S., dicarboxylic acid shuttle. C-6 to C-1 in the rabbit nerve cannot be taken as a direct measure of the extent of this reaction. If the citrate cleavage enzyme in the sciatic nerve of the rabbit is active in vivo, the loss of the radioactivity of C-6 in citrate can occur from the cleavages of citrate and through the dicarboxylic acid shuttle. Therefore, it is impossible to estimate the extent of C02 fixation at the oxalosuccinate level unless we know the activity of the citric cleavage enzyme in vivo. The ratio of radioactivity of C-6 to C-1 in the rabbit nerve damaged at desheathing, as shown by the significant decrease in the action potential, is approximately 1, and the ratio of the 2 carboxyl groups of aspartic acid is about 1 (Table 2). The specific activity of citric acid in damaged nerves is only 30 per cent of that found in intact rabbit nerves. These results strongly suggest that in the injured nerve the rate of carboxylation of a-ketoglutarate is significantly reduced. The carboxylation of a-ketoglutaric acid was investigated extensively by Ochoa and his colleagues1' in vitro, and recently the presence of the reaction was studied in the mammary glands in vitro,'2 in the perfused liver,'3 and in the brain in vivo (unpublished results). The rate of this reaction in the nerves of the lobster and rabbit indicates that the carboxylation of a-ketoglutaric acid may contribute to the control of the concentration of citric acid in the peripheral nerve. Furthermore, it seems of some interest to consider whether C02 fixation into a-ketoglutaric acid plays a significant role in nervous tissue by governing, through the citric acid cleavage enzyme,'4' 15 the acetyl CoA concentration and the level of acetylcholine. The major significance for rapid reaction in the nervous system may therefore lie with the cleavage enzyme and the formation of acetyl CoA as a source of acetylcholine. Since the rate of the carboxylation of a-ketoglutarate depends on the concentrations of a-ketoglutarate and C02,16 perhaps the high concentration of glutamic acid in the

5 Voi,. 54, 1965 BIOCHEMISTRY: WAELSCH ET AL nervous system acts as a metabolic buffer, as suggested many years ago" for controlling the concentration of a-ketoglutaric acid; the rate of carboxylation of this keto acid could thereby play a role in determining the rate of the Krebs cycle. In this connection it is of interest to remember that the effect of glutamic acid18 in acetylcholine formation by brain extract has been suggested to be due to citrate. 19 In this biochemical mechanism the effect of CO2 on specialized nervous structures (respiratory center and carotid body), as observed by the physiologists almost 100 years ago,' may find its explanation. Furthermore, the control of the citrate concentration may also explain the need of the peripheral nerves for CO2.2 Summary.-The rate of CO2 fixation into the intermediates of the citric acid cycle was measured in the lobster nerve and the effect of cations and drugs in the suspending medium was examined. If the extent of CO2 incorporation is taken as a measure of the rate of the citric acid cycle, it becomes apparent that the changes in the ionic flux through the nerve membranes produce a distinctive effect on the energy metabolism of the nerve. Citrate was isolated from lobster and rabbit nerves after incubation in C'4-bicarbonate Ringer's solution and the radioactivities of C-1 and C-6 were measured. It was found that CO2 fixation occurred at the oxajosuccinate and oxaloacetate levels. The biochemical significance of CO2 fixation, and especially the carboxylation of a-ketoglutarate in nervous tissue, is discussed. * This work was supported in part by grants from the National Institute of Neurological Diseases and Blindness (NB 00557), from the U.S. Air Force Office of Scientific Research, and by a contract between the Office of Naval Research and the Research Foundation for Mental Hygiene, Inc. (N. Y. State Psychiatric Institute Branch). For complete data on this work, see ref. 7. t U.S. Public Health postdoctoral fellow. t Fellow of the Rockefeller Foundation. ' Pfiuger, E., Arch. Physiol., 1, 61 (1868). 2 Lorente de N6, R., in Studies of the Rockefeller Institute (1947), vol Crane, R. K., and E. G. Ball, J. Biol. Chem., 189, 269 (1951). 4Ochoa, S., and E. Weisz-Tabori, J. Biol. Chem., 174, 123 (1948). 6 Berl, S., G. Takagaki, D. D. Clarke, and H. Waelsch, J. Biol. Chem., 237, 2570 (1962). 6 Waelsch, H., S. Berl, C. A. Rossi, D. D. Clarke, and D. P. Purpura, J. Neurochem., 11, 714 (1964). 7The data summarized in this manuscript are being published in detail in the papers submitted to J. Neurochem.: (a) Cote, L. J., S.-C. Cheng, and H. Waelsch; (b) Cheng, S.-C., and P. Mela, I.; (c) Cheng, S.-C., II.; (d) Naruse, H., S.-C. Cheng, and H. Waelsch; (e) Naruse, H., S.-C. Cheng, and H. Waelsch; (f) Naruse, H., S.-C. Cheng, and H. Waelsch. 8 Cheng, S.-C., and H. Waelsch, Biochem. Z., 338, 643 (1963). 9 Anfinsen, C. B., in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, 1955), vol. 1, p ' Whittam, R., and M. E. Ager, Biochem. J., 93, 1337 (1964). "lochoa, S., in The Enzymes, ed. Sumner and K. Myrback (New York: Academic Press, 1952), 1st ed., vol. 2, p Madson, J., S. Abraham, and I. L. Chaikoff, J. Biol. Chem., 239, 1305 (1964). 3 D'Adamo, A. F., Jr., and D. E. Haft, J. Biol. Chem., 240, 613 (1965). '4 Srere, P. A., and F. Lipmann, J. Am. Chem. Soc., 75, 4874 (1953). '5 Srere, P. A., Nature, 205, 766 (1965). '6 Ochoa, S., J. Biol. Chem., 114, 133 (1948). 17 Waelsch, H., Advan. Protein Chem., 6, 299 (1951). 18 Nachmansohn, D., H. John, and H. Waelsch, J. Biol. Chem., 150, 485 (1943). 19 Lipton, M. A., and E. S. G. Barron, J. Biol. Chem., 166, 367 (1946).

Biological oxidation II. The Cytric acid cycle

Biological oxidation II. The Cytric acid cycle Biological oxidation II The Cytric acid cycle Outline The Cytric acid cycle (TCA tricarboxylic acid) Central role of Acetyl-CoA Regulation of the TCA cycle Anaplerotic reactions The Glyoxylate cycle Localization