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. 1983 Jun;72(2):434–440. doi: 10.1104/pp.72.2.434

Transport of Dicarboxylic Acids in Castor Bean Mitochondria 1

Joseph Chappell 1,2, Harry Beevers 1
PMCID: PMC1066252  PMID: 16663021

Abstract

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of 14C from [14C]malate or [14C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of 14C from [14C]malate or [14C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent Km values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [14C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [14C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [14C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent Km of 2.5 millimolar and Vmax of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bowman E. J., Ikuma H. Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria: II. Role of Adenylates. Plant Physiol. 1976 Sep;58(3):438–446. doi: 10.1104/pp.58.3.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CANVIN D. T., BEEVERS H. Sucrose synthesis from acetate in the germinating castor bean: kinetics and pathway. J Biol Chem. 1961 Apr;236:988–995. [PubMed] [Google Scholar]
  3. Day D. A., Hanson J. B. Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria. Plant Physiol. 1977 Feb;59(2):139–144. doi: 10.1104/pp.59.2.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Douce R., Christensen E. L., Bonner W. D., Jr Preparation of intaintact plant mitochondria. Biochim Biophys Acta. 1972 Aug 17;275(2):148–160. doi: 10.1016/0005-2728(72)90035-7. [DOI] [PubMed] [Google Scholar]
  5. Johnson R. N., Chappell J. B. The transport of inorganic phosphate by the mitochondrial dicarboxylate carrier. Biochem J. 1973 Jul;134(3):769–774. doi: 10.1042/bj1340769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Journet E. P., Bonner W. D., Douce R. Glutamate metabolism triggered by oxaloacetate in intact plant mitochondria. Arch Biochem Biophys. 1982 Mar;214(1):366–375. doi: 10.1016/0003-9861(82)90041-8. [DOI] [PubMed] [Google Scholar]
  7. Jung D. W., Laties G. G. Citrate and succinate uptake by potato mitochondria. Plant Physiol. 1979 Apr;63(4):591–597. doi: 10.1104/pp.63.4.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LaNoue K. F., Schoolwerth A. C. Metabolite transport in mitochondria. Annu Rev Biochem. 1979;48:871–922. doi: 10.1146/annurev.bi.48.070179.004255. [DOI] [PubMed] [Google Scholar]
  9. Meijer A. J., Groot G. S.P., Tager J. M. Effect of sulphydryl-blocking reagents on mitochondrial anion-exchange reactions involving phosphate. FEBS Lett. 1970 May 11;8(1):41–44. doi: 10.1016/0014-5793(70)80220-4. [DOI] [PubMed] [Google Scholar]
  10. Mettler I. J., Beevers H. Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle. Plant Physiol. 1980 Oct;66(4):555–560. doi: 10.1104/pp.66.4.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Palmieri F., Prezioso G., Quagliariello E., Klingenberg M. Kinetic study of the dicarboxylate carrier in rat liver mitochondria. Eur J Biochem. 1971 Sep 13;22(1):66–74. doi: 10.1111/j.1432-1033.1971.tb01515.x. [DOI] [PubMed] [Google Scholar]
  12. Pfaff E., Klingenberg M., Ritt E., Vogell W. Korrelation des unspezifisch permeablen mitochondrialen Raumes mit dem "Intermembran-Raum". Eur J Biochem. 1968 Jul;5(2):222–232. doi: 10.1111/j.1432-1033.1968.tb00361.x. [DOI] [PubMed] [Google Scholar]
  13. Phillips M. L., Williams G. R. Anion transporters in plant mitochondria. Plant Physiol. 1973 Apr;51(4):667–670. doi: 10.1104/pp.51.4.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Phillips M. L., Williams G. R. Effects of 2-Butylmalonate, 2-Phenylsuccinate, Benzylmalonate, and p-Iodobenzylmalonate on the Oxidation of Substrates by Mung Bean Mitochondria. Plant Physiol. 1973 Feb;51(2):225–228. doi: 10.1104/pp.51.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Robinson B. H., Chappell J. B. The inhibition of malate, tricarboxylate and oxoglutarate entry into mitochondria by 2-n-butylmalonate. Biochem Biophys Res Commun. 1967 Jul 21;28(2):249–255. doi: 10.1016/0006-291x(67)90437-8. [DOI] [PubMed] [Google Scholar]
  16. Robinson B. H., Williams G. R. The sensitivity of dicarboxylate anion exchange reactions o transport inhibitors in rat-liver mitochondria. Biochim Biophys Acta. 1970 Aug 4;216(1):63–70. doi: 10.1016/0005-2728(70)90159-3. [DOI] [PubMed] [Google Scholar]
  17. Wiskich J. T. Phosphate-dependent Substrate Transport into Mitochondria: Oxidative Studies. Plant Physiol. 1975 Jul;56(1):121–125. doi: 10.1104/pp.56.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. von dem Hagen D., Löliger H. C. Zur Bedeutung Virus-neutralisierender Antikörper bei der Marek'schen Krankheit des Huhnes. 1. Mitteilung: Immunitätskontrolle nach Marek-Vakzination mit Puten-Herpes-Virus (PHV). Dtsch Tierarztl Wochenschr. 1978 Jun 5;85(6):220–222. [PubMed] [Google Scholar]

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