INHIBITION OF ACONITASE BY truns-aconitate*

Transcription

1 NHBTON OF ACONTASE BY truns-acontate* BY MURRAY SAFFRAN AND J. LEAL PRADO? (From the Departments of Biochemistry and Psychiatry, McGill University and the Allan Memorial nstitute of Psychiatry, Montreal, Canada) (Received for publication, June 27, 1949) Bernheim (2), studying the properties of the citric dehydrogenase system, noted that aconitic acid (presumably the trans isomer) inhibited the reduction of methylene blue by citric acid. More recently, Morrison and Still (3), in a short report, claimed that truns-aconitic acid inhibited competitively the reaction cis-aconitate c citrate, catalyzed by the aconitase of rhubarb leaf. The present studies show that trans-aconitate is a competitive inhibitor of aconitase of animal tissues and that trans-aconitate inhibits the respiration of tissue slices and causes the accumulation of citrate by surviving kidney cortex and liver slices. Methods Aconitase-The aconitase used in these studies was a water extract of pigeon breast muscle (4) prepared by homogenizing the muscle with water in a Waring blendor or in a glass homogenizer and centrifuging the homogenate at 3000 R.P.M. for 10 to 15 minutes. The cloudy supernatant, which contained most of the activity, was used. The activity of the enzyme preparation was approximately directly proportional to the mass of pigeon muscle extracted (Fig. 1). n all experiments the amount of enzyme is expressed in terms of the original weight of muscle. The breast muscle was stored in the freezing compartment of the refrigerator. The frozen tissue retained aconitase activity for many months (4). The extracts of muscle also retained aconitase activity if kept frozen, but lost activity slowly over a period of weeks, approximately half of the activity remaining after 21 days (Fig. 2). The extracts of frozen muscle exhibited no respiratory activity. Estimation of Citrate-The method of Natelson et al. (5), by which citric acid is estimated but not cis- or trans-aconitic or isocitric acids, was modified for use with the standard cuvettes of the Evelyn or Coleman universal photometers. * This investigation was carried out during the tenure of a Life nsurance Medical Research Fund Student Fellowship by one of us (M. S.) and with the aid of a grant from the National Research Council, Ottawa. A preliminary report of a portion of this investigation has been published (1). t Canada-Brazil Trust Fund Fellow. 1301

2 1302 NHBTON OF ACONTASE Manometric Methods-The studies on respiration were carried out with the usual Warburg technique in phosphate-buffered media. Chemical Preparations-trans-Aconitic acid was prepared from citric acid (6). cis-aconitic anhydride, which yields the acid when dissolved in water, was made from trans-aconitic acid (7). Other substances were purified commercial preparations. The acids were neutralized with equivalent amounts of sodium bicarbonate before use WEGHT OF MUSCLE N MG. AGE N DAYS FG. 1 FG. 2 FG. 1. Aconitase activity of an aqueous extract of frozen pigeon breast muscle. 10 PM of cis-aconitate in 1 ml. of 0.1 M phosphate buffer (ph 7.4) were incubated in open tubes with 1 ml. of an aqueous extract of 10 to 50 mg. of frozen tissue. The reaction was stopped at 30 minutes by the addition of 2 ml. of 25 per cent trichloroacetic acid and the contents of the tubes were analyzed for citrate. Temperature, 38. FG. 2. Stability of frozen aconitase extracts. Conditions the same as for Fig. 1. The extract in each determination represented 67 mg. of fresh muscle. Results nhibition of Aconitase-When cis-aconitate is incubated with aconitase, about 85 to 90 per cent is converted to citrate at equilibrium; citrate, incubated with aconitase, disappears to the extent of about 10 to 15 per cent (8). The addition of 0.06 M trans-aconitate to either of these systems diminishes the rate of interconversion. t also causes the reaction to stop before equilibrium is attained (Fig. 3). This may be the result of inactivation of the enzyme under the conditions of the experiment. The extent of the inhibition depends upon the concentration of transaconitate; increasing the concentration of trans-aconitate results in the decreased formation of citrate from cis-aconitate (Fig. 4). For a large portion of the curve the inhibition is a linear function of the common logarithm of the concentration of inhibitor.

3 M. SAFFRAN AND J. L. PRADO 1303 Nature of nhibition-prolonged exposure of aconitase to high concentrations of trans-aconitate (0.06 M) at 38 apparently results in inactivation of the enzyme, since the usual equilibrium is not attained (Fig. 3). The nature of the inhibition with lower concentrations of inhibitor was also investigated MNUTES FO. 3 FG. 4 FG. 3. nhibition of aconitase by trans-aconitate. ncubation carried out in open tubes containing 1 ml. of substrate (10 pm of citrate or cis-aconitate) in 0.1 M phosphate buffer (ph 7.4), either 1 ml. of buffer or 1 ml. of trans-aconitate (18Op~) in buffer, and 1 ml. of an aqueous extract of 30 mg. of frozen muscle. Temperature, 38. Reaction stopped by the addition of 2 ml. of a 25 per cent solution of trichloroacetic acid. 1 ml. aliquots of the supernatant were analyzed for citrate. FG. 4. nhibition of aconitase by increasing concentrations of trans-aconitate. ncubation carried out in Warburg flasks containing 0.1 ml. of cis-aconitate (10 PM), 2.0 ml. of trans-aconitate (5 to looopm), both in 0.1 M phosphate buffer (ph 7.4), and 0.5 ml. of aconitase extract (4 mg. of pigeon muscle) in buffer. The reaction was stopped after 60 minutes by the addition of 2.5 ml. of a 20 per cent solution of trichloroacetic acid and the contents of the flasks were analyzed for citrate. Gas, Nz; temperature, 37. Lineweaver and Burk (9) have developed graphic criteria for competitive inhibition: plotting the reciprocal of the initial concentration of substrate (l/so) against the reciprocal of the velocity of the reaction (l/v), in the presence of increasing amounts of a competitive inhibitor, produces a family of straight lines of increasing slope, but with a common intercept on the ordinate. Concentrations of cis-aconitate of to M were incubated for 10 minutes with aconitase and 0,0.005, and M trans-aconitate. Plotting l/so against l/v (Fig. 5) results in three straight lines of increasing slope, but with slightly different intercepts on the ordinate. The dif-

4 1304 NHBTON OF ACONTASE ference between the intercepts may be explained in part by experimental error, but it may be caused by a slight amount of the inactivation of the enzyme that was seen in Fig. 3, when higher concentrations of trans- /SO FG. 5 MlNUl-ES FG. 6 FG. 5. Competitive inhibition of aconitase by trans-aconitate. The numerals on the curves indicate the concentration of trans-aconitate in micromoles per ml. v = micromoles of citrate formed in 10 minutes; so = initial concentration of cis-aconitate in micromoles per ml. The experiment was performed in open tubes containing 0.1 to 2.0 ml. of cis-aconitate (1 to 20 PM) in 0.1 M phosphate buffer (ph 7.4), 1 or 2 ml. of trans-aconitate (50 or 100 PM) in buffer, buffer to 7.0 ml., and 3.0 ml. of an aqueous extract of frozen pigeon muscle, representing 20 mg. of tissue. Temperature, 38. The reaction was stopped after 10 minutes by the addition of 1 ml. of a 100 per cent (weight by volume) solution of trichloroacetic acid and the contents of the tubes were analyzed for citrate. FG. 6. nhibition of the respiration of rat kidney cortex slices by trans-aconitate and malonate. Curves A and B, normal respiration; Curve C, M trans-aconitate; Curve D, M malonate; Curve E, nr trans-aconitate together with M malonate; Curve F, 0.02 M Pans-aconitate; Curve G, 0.02 M malonate; Curve H, 0.02 M Pans-aconitate together with 0.02 M malonate. Medium, 2 ml. of calcium-free Krebs-Ringer-phosphate solution. Gas, 02; temperature, 37. aconitate were used. With lower concentrations of inhibitor the inhibition appears to be predominantly competitive. nhibition of Respiration-The addition of trans-aconitate to slices of kidney cortex or liver, respiring in calcium-free Ringer-phosphate medium, caused a definite decrease in the rate of oxygen consumption. The in-

5 &. SAFFRAN AND J. L. PRADO 1305 ADDED&f&t-RATE. ' il NONE p-?mq /&g 1 MALATE FUMARATE CTRATE &ACONTATE 0 5 O FG. 7. Effect of the addition of compounds of the Krebs cycle on the inhibition of the respiration of slices of rat kidney cortex by trans-aconitate. Unshaded bars, no inhibitor added; shaded bars, respiration in the presence of the indicated concentration of trans-aconitate. Conditions the same as in Fig. 6. TABLE E,fect of ncreasing Concentrations of trans-aconitate on Respiration of Tissue Slices in Calcium-Free Ringer-Phosphate Solution Rabbit Rat kidney liver kidney cortex cortex trans-aconitate N x lo added - - li LM per 100 mg. per hr. Oxygen uptake nhibition )er ten hibition of respiration of rat kidney cortex slices by and 0.02 M trans-aconitate is compared with the inhibition by the same concentrations of malonate in Fig. 6. The inhibition of respiration was also seen

6 1306 NHBTON OF ACONl ASE in the presence of added malate or fumarate, but was largely overcome by the addition of the tricarboxylic acids, citric and cis-aconitic (Fig. 7). The inhibition of respiration of slices of various tissues is increased by increasing concentrations of trans-aconitate (Table ). E$ect on Accumulation of Citrate-The accumulation of citrate by slices of kidney cortex and liver was greatly increased in the presence of 0.02 M trans-aconitate. This effect of truns-aconitate was enhanced by the addition of 0.01 M pyruvate or malate, but not by the addition of acetate. The results obtained with rabbit kidney cortex slices are illustrated in Fig SO SO 120 TME N MNUTES FG. 8. Effect of trans-aconitate on the accumulation of citrate by slices of rabbit kidney cortex. 0, no inhibitor added; l,0.02 M trans-aconitate added. Medium, 2 ml. of calcium-free Krebs-Ringer-bicarbonate solution, ph 7.4, with an atmosphere of 5 per cent COz in OZ. Temperature, 37. Tissue killed by the addition of 0.5 ml. of a 100 per cent (weight by volume) solution of trichloroacetic acid and the contents of the flasks analyzed for citrate. DSCUSSON Research in intermediary metabolism has elucidated many of the reactions in the Krebs or tricarboxylic acid cycle, but several gaps remain. One of the unknown regions is that involving the tricarboxylic acids, citric, cis-aconitic, and isocitric. Evidence from isotope studies (10-12) seemed to eliminate symmetrical compounds, like citrate, from the direct path of the Krebs cycle, and consequently citrate was placed in a side reaction. However, recent theoretical considerations by Ogston (13) have pointed out that the formation of only one optical isomer of isocitrate from citrate indicates that citrate is attached to aconitase in one way only, in effect endowing the molecule with asymmetrical properties. This argument has been strengthened by experimental evidence. Stern and Ochoa (14) reported recently that citrate was formed from oxalacetate and acet.ate under conditions in which aconitase could not be demonstrated,

7 M. SAFFFCAN AND J..,. PRADO 1307 Another experimental approach to this problem is made possible by using trans-aconitate as an inhibitor of aconitase. f it is assumed that one of the three tricarboxylic acids, citric, cis-aconitic, or isocitric, is the product of the condensation of oxalacetate and a a-carbon compound, three schemes can be set up. f the additional assumption is made that trans-aconitate specifically inhibits aconitase (indicated by the dashed lines in the schemes), the effect of the addition of trans-aconitate on the accumulation of citrate would depend upon the order in which the tricarboxylic acids are formed. Scheme (A) (B) (Cl Citrats+G-Aconitate&socitrate citrate *iconitate* -- \ -- t Citrate de-aiionltate C socitrata sooitrate i Krebs cycle t 4 Krebe cycle Krebs 4 cyole A would result in the accumulation of more citrate in the presence than in the absence of the inhibitor, because the breakdown of citrate would be inhibited. Scheme B would result in the accumulation of less citrate because its formation would be inhibited. Scheme C would drastically reduce the amount of citrate formed and would not explain the inhibition of respiration by truns-aconitate. n this investigation citrate accumulated in larger amounts when transaconitate was added to tissue slices, suggesting that Scheme A is probably involved. However, similar results might be expected from Scheme B if

8 1308 NHBTON OF ACONTASE trans-aconitate inhibits the reaction cis-aconitate $ isocitrate more strongly than the reaction cis-aconitate Z$ citrate, implying the existence of the two aconitases of Jacobson et al. (15). Alternatively, these results could be explained by all three schemes if the further breakdown of isocitrate were inhibited more strongly by trans-aconitate than the reactions of the aconitase system (Scheme D). Experiments to test the effect of trans-aconitate on the reaction eisaconitate G isocitrate and on the breakdown of isocitrate are now in progress. We are indebted to Dr. K. A. C. Elliott and Dr. 0. F. Denstedt for their interest and encouragement in this investigation. (D) SUMMARY 1. The inhibition of aconitase by trans-aconitate is primarily competitive. Prolonged exposure to high concentrations of inhibitor apparently results in inactivation of the enzyme. 2. The respiration of kidney cortex and liver slices is inhibited by transaconitate. This inhibition is largely overcome by the addition of citrate or cis-aconitate. 3. The accumulation of citrate by tissue slices is markedly increased by trans-aconitate, suggesting that citrate is formed by the condensation of oxalacetate and a a-carbon compound. 4. The accumulation of citrate from acetate by slices of rabbit kidney cortex is much less than from pyruvate or malate. Xrebs cycle BBLOGRAPHY 1. S&ran, M., and Prado, J. L., Federation Proc., 7,182 (1948). 2. Bernheim, F., Biochem. J., 22, 1178 (1928). 3. Morrison, J. F., and Still, J. L., Australian J. SC., 9,150 (1947). 4. Krebs, H. A., and Eggleston, L. V., Biochem. J., 38,426 (1944). 5. Natelson, S., Lugovoy, J. K., and Pincus, J. B., J. Biol. Chem., 170,597 (1947). 6. h-0. Suntheses. toll. 2, 12 (1943).

9 M. SAFFRAiV AND J. L. PRADO Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and related methods for the study of tissue metabolism, Minneapolis, 185 (1945). 8. Martius, C., and Leonhardt,., 2. physiol. Chem., 278,208 (1943). 9. Lineweaver, H., and Burk, D., J. Am. Chem. Sot., 66,658 (1934). 10. Evans, E. A., Jr., and Slotin, L., J. Biol. Chem., 141,439 (1941). 11. Wood, H. G., Werkman, C. H., Hemingway, A., and Nier, A. O., Chem., 142, 31 (1942). 12. Weinhouse, S., and Millington, R.., J. Am. Chem. Sot., 69,3089 (1947). 13. Ogston, A. G., Nature, 162, 963 (1948). 14. Stern, J. R., and Ochoa, S., J. Biol. Chem., 179,491 (1949). 15. Jacobson, K. P., Soares, M., and Tapadinhas, J., Bull. Sot. chim. biol., 22, 48 (1940).

10 NHBTON OF ACONTASE BY trans -ACONTATE Murray Saffran and J. Leal Prado J. Biol. Chem. 1949, 180: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at html#ref-list-1