OXIDATIVE PHOSPHORYLATION PROCESSES IN NUTRITIONAL MUSCULAR DYSTROPHY*

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1 OXIDATIVE PHOSPHORYLATION PROCESSES IN NUTRITIONAL MUSCULAR DYSTROPHY* BY J. P. HUMMELt (From the Biochemical Laboratory, State University of Iowa, Iowa Citg) (Received for publication, October 9, 1947) Nutritional muscular dystrophy is characterized by a pronounced loss of contractile power which appears to precede the degeneration of the muscle fibers (1). This paralysis may be due either to an impairment of the contractile mechanism as evidenced by the disappearance of the cross striations or to an impairment in energy utilization. Since the energy for muscle contraction is thought to be mobilized by oxidative phosphorylation mechanisms, this study was undertaken to discover whether any such processes may be affected in dystrophy, specifically the phosphorylation of creatine associated with the oxidation of carbohydrate. If the oxidation of cuglycerophosphate and fructosel, 6diphosphate is assumed to proceed entirely through the so called MeyerhofEmbden Parnas pathway (2) to pyruvic acid, the transfer of hydrogen from the substrate at each oxidative step may proceed either aerobically through the coenzyme Iflavincytochrome systems to molecular oxygen or anaerobically by means of the coenzyme Ilinked dismutation with pyruvic acid to form lactic acid. Two energyrich phosphate bonds, transferable to creatine through the adenylic system, would be formed per mole of triose phosphate oxidized to pyruvic acid, but only one would be formed under fluoride inhibition. Phosphorylation was therefore studied by measurement of the phosphocreatine formed from creatine, and oxidation by the oxygen consumed and lactic acid formed. EXPERIMENTAL Muscular dystrophy was produced in hamsters by the same procedure as previously described (3). It was also produced in guinea pigs placed on the same diet, with the oral administration of 10 mg. of ascorbic acid every other day. In this species, dystrophy became acute after about 5 weeks and was characterized by a very severe paralysis, loss in weight, and sudden death. Control animals, maintained on the same diet, were given ascorbic acid as above and 15 mg. of tocopherol acetate1 every 4 days. No symp * Aided by a grant from the John and Mary R. Markle Foundation. t Present address, WennerGrens Institute, University of Stockholm, Stockholm, Sweden. 1 Kindly supplied by HoffmannLa Roche, Inc. 421

2 422 OXIDATIVE PHOSPHORYLATION IN DYSTROPHY toms of the guinea pig wriststiffness described by van Wagtendonk and Wulzen (4) were observed. The method of preparing distilled water homogenates of thigh muscle has been described (3). Each Warburg flask contained, besides the indicated substrate in the side arm, the following in micromoles: crcatine 230, potassium chloride 400, magnesium chloride 20, nicotinamide 20, coenzyme I 0.75, adenosine triphosphate 1.33, and sodium phosphate buffer (ph 7.4) 10, and 100 mg. of fresh muscle (as 10 per cent muscle homogenate) in a total volume of 3.0 ml. The center well contained 0.1 ml. of 2 N sodium hydroxide. Tn the aerobic experiments, each flask also contained 8 X 10s moles of cytochrome c in an air atmosphere. In the anaerobic experiments, each flask contained 10 micromoles of lithium pyruvate2 under a nitrogen atmosphere. In certain experiments, 5 micromoles of sodium fluoride were added to inhibit the utilization of 2phosphoglyceric acid. Cytochrome c was prepared according to the method of Keilin and Hartree (5), as modified by Potter (6). Coenzyme I of about 40 per cent purity was prepared from starchfree bakers yeast by the method of Williamson and Green (7). The barium salt of adenosine triphosphate (ATP) was prepared by the method of Needham (8), as modified by DuBois, Albaum, and Potter (9). The product was 91 per cent pure, as measured by 7 minute hydrolysis. All additions were adjusted to ph 7.4 and employed at concentrations optimum for maximum phosphorylation. *411 incubations were conducted at 37. Manometric readings were taken 20 minutes after the substrate was tipped in from the side arm. The flask contents were then quickly chilled to 0 and deproteinized with trichloroacetic acid, according to the procedure outlined by Potter (10). The neutralized filtrate was analyzed for phosphocreatine by an adaptation (10) of the method of Fiske and Subbarow (ll), and for triose phosphate by 30 minute hydrolysis in 1 N sodium hydroxide at 25, followed by the usual calorimetric determination for inorganic phosphate. Lactic acid in the filtrate was determined by the procedure of Barker and Summerson (12). The data reported are net changes obtained by subtracting the values for control flasks which lacked only substrate. They are expressed as micromoles per hour, except those for oxygen consumption, which, in order to be on the same basis as lactic acid data, are expressed as microatoms per hour. In computing phosphocreatine to oxygen ratios (P to oxygen), the corrcsponding anaerobic phosphocreatine to lactic acid rat,io (P to lactic) is assumed to prevail under aerobic conditions. Therefore (PA (lactic,) X ((P8,,,A : (Iacticanser.)) P:oxygen = ~ (Oxygen) 2 Generously furnished by Dr. R. M. Featherstone of the Department of Pharmscology.

3 J. P. HUlMMEL 423 RESULTS AND DISCUSSION With normal guinea pig muscle and glycerophosphate as substrate, as shown in Table I, the addition of fluoride reduced the formation of lactic acid and phosphocreatine by about half, although the oxygen consumption was only slightly diminished. The P to oxygen ratio is ( X 0.45)/6.0 or 0.77 in the absence of fluoride, and by similar calculation it is 0.70 in the presence of fluoride. According to theory, the former value is too small, due probably to the leak of energyrich bonds by ATPase action. Under anaerobic conditions, however, less than half a mole of phosphocreatine was formed per mole of lactic acid, without fluoride, and even less in its presence. The TABLE Phosphorylation of Creatine from Oxidation of Glycerophosphate by Guinea Pig Muscle Homogenate* Oxygen uptake is expressed as microatoms per hour. Lactic acid, phosphocreatine, and triose phosphate are expressed as micromoles per hour. The ratios in the last two columns are explained in the text. Phosphc Triose Condition Gas Fluoride :g:: 2 creatine >hosphatc P:lactic : oxygen formed formed formed Normal (7 ani 0: mals) I N: Dystrophic ( animals) N I * Each flask contained the reaction mixtures described in the text, with 100 micromoles of sodium glycerophosphate (Eastman) as substrate. I further oxidation of pyruvic acid may account in part for the lack of agreement between the aerobic and anaerobic phosphorylation ratios. In the presence of fluoride, however, this disparity demands a different explanation; in such cases, the phosphorylation efficiency may possibly depend upon the potential gradient through which the hydrogen transport system is operative (13). The differences between normal and dystrophic guinea pig muscle are striking. In dystrophic muscle, the aerobic formation of lactic acid and phosphocreatine was very small, although the oxygen consumption was equal to that of normal muscle. This is reflected in the sharp reduction of the P to oxygen ratios in the case of dystrophic muscle. Under anaerobic

4 421 OXJDATIVE PHOSP~ORYLATION IN DYSTROPHY conditions, lactic acid was less than in the normal, but the efficiency of oxidative phosphate transfer was markedly diminished, as judged by the much smaller P to lactic ratio. Addition of fluoride lowered this still further. In all cases, the amounts of triose phosphate formed were small, but the slight accumulation with dystrophic muscle may be significant. Normal hamster muscle produced much less phosphocreatine than normal guinea pig muscle, although the oxygen consumption was very much greater. As seen in Table II, oxygen consumption and lactic acid and phosphocreatine formation were all diminished in dystrophic hamster muscle. Addition of fluoride lowered the phosphocreatine synthesis to almost zero without noticeably affecting either the aerobic or anaerobic Condition Normal mals) TABLE Phosphoryktion of Creatine from Oxidation of Glycerophosphate by Hamster Muscle Homogenates* (6 ani Dystrophic (5 animals) 02 N2 I 02 I N2 I Fluoride :g:lz II y;?tj formed e I!:lactic _ ^ P:oxygen * Each flask contained the reaction mixture described in the text, with 100 micromoles of sodium glycerophosphate as substrate. The data are expressed in the same terms as those used in Table I. oxidations. As with guinea pig muscle, triose phosphate values were small, but there appeared to be a slight accumulation with dystrophic muscle. The oxidation of fructosel,6diphosphate presents a somewhat different picture. With guinea pig muscle, as shown in Table III, fluoride inhibited the formation of lactic acid and phosphocreatine under aerobic and anaerobic conditions with both normal and dyst#rophic muscle, but stimulated the oxygen uptake, probably by preventing the formation of pyruvate which would act as a hydrogen acceptor. A disparity between the aerobic and anaerobic phosphorylation, similar to that seen with glycerophosphate oxidation, also exists with normal fructosel,6diphosphate oxidation. The differences between normal and dystrophic guinea pig muscle were not as great with fructosel,6diphosphate as with glycerophosphate as substrate. Under aerobic conditions, lactic acid and phosphocreatine were

5 3. P. HUMMEL 425 somewhat diminished in dystrophy, but oxygen uptake was unaltered. A comparison of the P to oxygen ratios, however, shows that dystrophy sig TABLE Phosphorylation of Creatine from Oxidation of Ffuctosel,&diphosphate by Guinea Pig Muscle Homogenate* Phospho Triose Condition Gas Fluoride creatine P Ihosphatl :&tic P :o?lygen formed formed. _ Normal mals) (6 ani Dystrophic (5 animals) 02 N2 02 N2 I III * Each flask contained the reaction mixture described in the text, with 30 micromoles of fructosel, 6diphosphate (Schwartz) as substrate. The data are expressed in the same terms as those used in Table I. Condition Normal mals) TABLE Phosphorylation of Creatine from Oxidation of Fructose1,6diphosphate by Hamster Muscle Homogenate* (5 ani Dystrophic (5 animals Gas Fluoride,: t 12.2 N N2 I + IV WC formed Phosphocreatine formed P:oxygen * Each flask contained the reaction mixture described in the text, with 30 micromoles of fructose1,6diphosphate as substrate. The data are expressed in the same terms as those used in Table I. nificantly reduced the aerobic phosphorylation efficiency. Under anaerobic conditions, the P to lactic ratios were in no way diminished in dystrophy. The oxidation of fructosel,bdiphosphate by hamster muscle, as shown in Table IV, was similar to that by guinea pig muscle, except that oxygen consumption, lactic acid, and phosphocreatine were all diminished in

6 426 OXiDATlVE PifO& HORYLATIOPj fnf DYSTROPHY dystrophy, with the result that changes occur in the phosphorylation efliciencies which are difficult to evaluate. Under anaerobic conditions, the formation of lactic acid by dystrophic muscle was very much smaller, although surprisingly the phosphocreatine formation was not greatly different from the normal. Since, with dystrophic guinea pig muscle, neither the oxygen uptake nor the anaerobic formation of lactic acid was greatly altered from the normal, it follows that the oxidations to the 1,3diphosphoglyceric acid step are probably not impaired in dystrophy. In the case of glycerophosphate, however, the subsequent phosphorylation steps do appear to be impeded, the evidence being the lowered phosphocreatine formacon, and, in the aerobic oxidation without fluoride, the lowered lactic acid formation. This lactic acid could come only from the pyruvic acid produced. If the slightly increased triose phosphate values have significance, this is additional evidence of such a barrier. Disturbances in phosphorylation may be general in the oxidative metabolism of dystrophic muscle. Lu et al. (14) found that the ability of dystrophic muscle to phosphorylate glycogen was reduced by half. Royer (15) similarly found that dystrophic muscle lost the ability to derive phosphate bond energy from the oxidation of certain members of the tricarboxylic acid cycle. His data indicated that there was no impairment in the transfer of energyrich phosphate bonds from enolphosphopyruvic acid to creatine in dystrophy. The differences between the phosphorylation efficiencies of fructosel,6 diphosphate and glycerophosphate are not explained if both are oxidized by the same pathway in the guinea pig. The reason for the differences in their oxidation is more apparent; 1 mole of fructosel, 6diphosphate yields 2 moles of pyruvic acid; 1 mole of glycerophosphate yields only 1 mole of pyruvic acid. Therefore, if primed by a very small amount of pyruvic acid, the anaerobic oxidation of fructosel,6diphosphate may theoretically proceed entirely to lactic acid, according to the well known mechanism for anaerobic glycolysis. Since, however, glycerophosphate requires two dehydrogenation steps, at least half of the oxidation must proceed aerobically unless an excess of pyruvic acid is already present. Thus the greater tendency of fructosel,6diphosphate oxidation to proceed anaerobically may be explained. The large accumulation of triose phosphate with fructosel.,6diphosphate as substrate probably results from the high activity of muscle aldolase (16). The greatly increased triose phosphate values with dystrophy may be due either to a barrier in the system or to an increased aldolase activity. With glycerophosphat,e, triose phosphate formation is limited by the rate of the initial oxidation step and thus accumulates in small amounts.

7 J. P. HUMiiEL 427 Dystrophy in hamsters impeded both oxidation and phosphorylation in the utilization of either substrate; oxygen uptake, lactic acid, and phosphocreatine formation were all diminished. These alterations would seem to be an indirect or subsidiary effect of the avit,aminosis because the in vitro addition of octocopherol phosphate did not restore normal activity to dystrophic muscle homogenates. Furthermore, in severely dystrophic rabbit muscle, a similar impairment in oxidative phosphorylation could not be demonstrated. It would seem that the action of tocopherol in vivo is mediated through as yet unrecognized agencies which are lost in vitro, and which may vary from species to species. These species differences make it difficult to generalize about the metabolic disturbances encountered in nutritional muscular dystrophy. TABLE Adenosinetriphosphatase Activity of Normal and Dystrophic Hamster and Guinea Pig Muscle* Animal ca++ Normal Dystrophic Hamster Guinea pig * Expressed as y of P split from ATP per mg. of tissue (wet, weight) in 15 minutes at 37, ph 7.4, corrected for phosphate content of the tissue and for nonenzymatic ATP hydrolysis. Finally, in a comparison of normal and dystrophic muscle, it must be emphasized that there is a smaller active mass of muscle as the result of necrosis and fibrotic infiltration which may, in part, explain the lowered enzymatic activity in advanced dystrophy. There was a fair parallelism between the lowered oxidative phosphorylation and the muscle degeneration observed histologically in sections of the same muscle.3 As possible indices of cellular destruction, assays of ATPase activity (adenylpyrophosphatase (17)) according to the micromethod of DuBois and Potter (18) were carried out in triplicate on the same tissue homogenates as were used in the phosphorylation experiments. Although the method may provide only an approximation, an appreciable destruction of RTPase had taken place in dystrophic muscle, as shown in Table V. The activation of ATPase by the high calcium content of dystrophic muscle has been suggested by Morgulis and Jacobi (19) as a cause for the high respiration through the destruction of ATP and the accompanying 3 Sections were made by the Department of Pathology. V

8 428 OXIDATIVE PHOSPHORYLATION IN DYSTROPHY increase in inorganic phosphate. In our hands, M calcium chloride, sufficient to give complete activation, stimulated the ATPase of normal and dystrophic muscle to the same extent. The extra calcium of dystrophic muscle is probably unionized and incapable of stimulating ATPase. Since ATPase is believed to be intimately associated with myosin (20), dystrophy appears to involve an alteration of the contractile structure of the muscle as well as an impairment in the utilization of energy for contraction. The author gratefully and G. Kalnitsky. acknowledges the helpful criticisms of H. A. Mattill SUMMARY The phosphorylation of creatine by normal and dystrophic hamster and guinea pig muscle homogenates was studied in the aerobic and anaerobic oxidation of glycerophosphate and fructosel, 6diphosphate in the presence of the necessary known cofactors. With glycerophosphate as substrate, the aerobic and anaerobic oxidation rate by dystrophic guinea pig muscle was little different from the normal, but the coupled phosphorylation of creatine was greatly diminished. With fructose1,6diphosphate, the effects of dystrophy were similar but less marked. Dystrophy appeared to affect the aerobic phosphorylation more than the anaerobic. In dystrophic hamster muscle homogenates, both oxidation and phosphorylat.ion processes were impeded. These alterations appear, to be indirect effects of the vitamin E deficiency, but may partially explain the associated paralysis. Dystrophy in hamsters and guinea pigs markedly lowers the muscle adenosinetriphosphatase activity and is interpreted to mean that the contractile structure in dystrophic muscle is also impaired. BIBLIOGRAPHY 1. Knowlton, G. A., and Hines, H. M., Proc. Sot. Exp. Biol. and Med., 38, 665 (1938). 2. Meyerhof, O., in Biological symposia, Lancaster, 6, 141 (1941). 3. Basinski, D. H., and Hummel, J. P., J. Biot. Chem., 167,339 (1947). 4. van Wa.gtendonk, W. J., and Wulzen, R., Arch. Biochem., 1,373 (1943). 5. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 122, 298 (1937). 6. Potter, V. R., in Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and related methods for the study of tissue metabolism, Minneapolis, 188 (1945). 7. Williamson, S., and Green, D. E., J. Biol. Chem., 136,345 (1940). 8. Needham, D. M., Biochem. J., 36, 113 (1942). 9. DuBois, K. P., Slbaum, H. G., and Potter, V. R., J. Biol. Chem., 147,699 (1943). 10. Potter, V. R., Arch. Biochem., 6,439 (1945). 11. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 81,657 (1929).

9 J. P. HTJMMEL Barker, S. B., and Summerson, W. H., J. Biol. Gem., 138,535 (1941). 13. Ochoa, S., Ann. New York Acad. SC., 47,835 (1947). 14. Lu, G. D., Emerson, G. A., and Evans, H. M., Am. J. Physiol., 129,408 (1940). 15. Boyer, P. D., Thesis, University of Wisconsin (1943). 16. Meyerhof, O., and Lohmann, K., Biochem. Z., 2 71, 89 (1934). 17. Moog, F., and Steinbach, II. B., J. Cell. and C omp. Physiol., 25, 133 (1945). 18. DuBois, K. P., and Potter, V. R., J. BioZ. Chem., 150,135 (1943). 19. Morgulis, S., and Jacobi, H. P., Quart. Bull. Northwestern Univ. Med. School, 20, 92 (1946). 20. Engelhardt, W. A., and Ljubimowa, M. N., Nature, 144, 668 (1939).

10 OXIDATIVE PHOSPHORYLATION PROCESSES IN NUTRITIONAL MUSCULAR DYSTROPHY J. P. Hummel J. Biol. Chem. 1948, 172: 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 tml#reflist1

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