of Succinic Dehydrogenase 3-Nitropropanoate’ J. W.
Hawaii Agricultural Honolulu, Hawaii
The component of Zndigojera endecaphylla Jacq. which is toxic to fowl (Rosenberg and Palafox, 1950) and mammals (Emmel and Ritchey, 1941) has been indentified as 3-nitropropanoic acid (3-NPA) (Morris et al., 1954). Intravenous or intraperitoneal administration results in methemoglobinemia in rats (Matsumoto et al., 1961). Scott (1943) has shown that nitrite ion is produced when nitroethane is metabolized by rabbits, and presumably some nitrite is formed from 3-NPA by an analagous process. However, the amounts of methemoglobin produced by lethal dosages of 3-NPA are too low to account for the fatal effects. The observed mortality could be explained if 3-NPA inhibits some enzyme system of vital importance. This report details the results of an investigation into the effects of 3-NPA on the activity of one such enzyme, succinic dehydrogenase from rat heart muscle. EXPERIMENTAL
Enzyme preparation. Succinic dehydrogenase was prepared from rat heart muscle by the method of Keilin and Hartree (1947). Fat-free dry weights were determined by the procedure of Slater (1949). Enzyme assay. The spectrophotometric procedure of Slater and Bonner (1952), as described in detail by Thorn ( 1953)) was used except that measurements were made at 400 mp with a Beckman model B spectrophotometer, Unfortunately, the more specific method of Singer and Kearney (1957) was not applicable due to the reaction of 3-NPA with phenazine methosulfate to yield an inactive green dye (J. W. Hylin and J. Padayhag, unpublished observations). Reagents. All reagent solutions were prepared from chemicals of analytical reagent grade. Glass distilled water was used throughout. Solutions of malonic acid, succinic acid, and 3-NPA were adjusted to pH 7.3 with NaOH. RESULTS
Preliminary experiments showed 3-NPA to be an inhibitor of succinic dehydrogenase from rat heart muscle. The following experiments were performed to elucidate the nature of this inhibition. Malonic acid was incorporated into the experimental design as a reference inhibitor. The effects of varying concentrations of 3-NPA on enzyme activity were compared with similar effects caused by malonate (Fig. 1). The amount 1 Published as Technical
with the approval Paper No. 530.
of the Director
of the 168
of inhibition, in both cases, is directly proportional to the concentration of inhibitor. The effect of substrate concentration on enzyme activity in the presence of a constant amount of inhibitor was next investigated. The data, plotted by the method of Line7
INHIBITOR MOLARITY FIG. 1. Effect of inhibitor concentration on the activity of succinic dehydrogenase in the presence of a constant amount of substrate. V, maximum velocity; V, observed velocity; 0, malonate; l , 3-NPA. Activity measured in the presence of borate, pH 7.3, 0.1 44; succinate, 0.020 &f; K,Fe(CN),, 0.001 M; KCN, 0.01 M; enzyme preparation, 0.30 mg/ml.
weaver and Burk (1934) (Fig. 2), show that 3-NPA is a competitive inhibitor of succinic dehydrogenase. Km, Kimalonate, Ki3.~pA,and Km/Ki for the two inhibitors were calculated using these results (Table 1). Good agreement was obtained when the TABLE COMPARISON
x lo-GM -
values were compared with those of Thorn (1953). When the amount of enzyme preparation was varied, the inhibition of enzyme activity was found to be a linear function of the enzyme concentration (Fig. 3). This is added proof for the competitive nature of the inhibition.
I I I I I I X
loo; :-:’ SO
2oC 00 0
FIG. 2. Competitive inhibition of succinic dehydrogenase by malonate and 3-NPA. s, Molar concentration of succinate; v, change in absorption per minute; 0, no inhibitor; 0, 1 X IO-sM malonate; 61 4 X lo--41b’ 3-NPA; X, 8 X 10-4&f 3-NPA. Activity measured in the presence of borate, pH 7.3, 0.1 M; K,Fe(CN)o, 0.001 M; 0.01 h4; enzyme preparation, 0.42 mg/ml.
0.04 CONCENTRATION (mg.fot-free
0.06 OF ENZYME dry
FIG. 3. Effect of enzyme concentration on the inhibition in absorption; 0, no inhibitor; 0, 5 X IO-“M malonate; ured in the presence of borate, pH 7.3, 0.1 M; succinate,
L 0. 16
of succinic dehydrogenase. A A, change 0, 4.3 X 10-a 3-NPA. Activity meas0.020 M; KaFe(CN)s, 0.001 M; KCN,
Inhibition of succinic dehydrogenaseby 3-NPA was not unexpected. Examination of the chemical structure of 3-NPA suggestsits similarity to succinic acid. The values given in Table 1 indicate that 3-NPA is a, relatively poor inhibitor when compared with malonate. Thorn (1953) has discussedthe problems inherent in the determination of the kinetic constants for succinic dehydrogenase.Nevertheless, the good agreement between the experimentally determined values for Km and Kimalonate and those reported by Thorn, gives a measureof the reliability of the values for Ki3.NPA.The enzyme used in this investigation was from a source different from that used by Thorn. However, Singer et al. (1956) have pointed out that the Km for succinic dehydrogenasefrom different sourcesis remarkably constant. Detection of the inhibition of succinic dehydrogenaseby 3-NPA in vivo was attempted. Enzyme activities in heart muscle and brain tissue from 3-NPA poisoned rats did not differ significantly from the activities found in untreated animals. Therefore, the contribution of this weak competitive inhibition to the mortality of animals treated with 3-NPA cannot be assessedat this time. SUMMARY 3-Nitropropanoate (3-NPA) has been shown to be a competitive inhibitor of rat heart muscle has been determined to be 1.84-2.0X succinic dehydrogenase. The dissociation constant Ki,.,,, 10-4. 3-NPA is approximately one-twentieth as powerful an inhibitor of this enzyme as malonate. REFERENCES M. W., and RITCHEY, G. E. (1941). The toxic constituent of Indigofera endecaphylla Jacq. for rabbits. Agron. J. 22, 675-677. KEILIN, D., and HARTREE, E. F. (1947). Activity of the cytochrome system in heart-muscle preparations. Biochem. J. 41, 500-502. LINEWEAVER, H., and BURK, D. (1934). Determination of enzyme dissociation constants. J. Am. EMMEL,
H., HYLIN, J. W., and MIYAHAR~, A. (1961). Methemoglobinemia in rats injected with 3-nitropropanoic acid, sodium nitrite, and nitroethane. Toxicol. Appl. Pharmacol. 3, 493-499. MORRIS, M. P., PAGAN, C., and WARMKE, H. E. (1954). Hiptagenic acid, a toxic component of Indigofera endecaphylla. Science 119, 322-323. ROSENBERG, M. M., and PALA~OX, A. L. (1950). The effect of creeping indigo (Indigofera endecaphylla) when fed to growing chickens. World’s Poultry Sci. J. 6, 284-291. SCOTT, E. W. (1943). The metabolism of mononitroparaffins. III. The concentration of nitroethane, nitrite and nitrate in the blood of rabbits during exposure by inhalation and oral administration. J. Ind. Hyg. Toxicol. 25, 20-25. SINGER, T. P., and KEARNEY, E. B. (1957). Determination of succinic dehydrogenase activity. MATSUMOTO,
T. P., KEARNEY, E. B., and BF.RNATH, P. (1956). Studies on succinic dehydrogenase. II. Isolation and properties of the dehydrogenase from beef heart. J. Biol. Chem. 223, 599-613. SLATER, E. C. (1949). A com’parative study of succinic dehydrogenase-cytochrome system of heart-muscle and in kidney. Biochem. J. 46, l-8. SLATER, E. C., and BONNER, W. D. (1952). The effect of fluoride on the succinic oxidase system. Biochem. J. 52, 185-196. THORN, M. B. (1953). Inhibition by malonate of succinic dehydrogenase in heart-muscle preparations. Biochem. J. 54, 540-547. SINGER,