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. 1999 Sep 15;342(Pt 3):537–544.

Defective kinetics of cytochrome c oxidase and alteration of mitochondrial membrane potential in fibroblasts and cytoplasmic hybrid cells with the mutation for myoclonus epilepsy with ragged-red fibres ('MERRF') at position 8344 nt.

H Antonická 1, D Floryk 1, P Klement 1, L Stratilová 1, J Hermanská 1, H Houstková 1, M Kalous 1, Z Drahota 1, J Zeman 1, J Houstek 1
PMCID: PMC1220494  PMID: 10477264

Abstract

We have investigated pathogenic effects of the tRNA(Lys) A8344G mutation associated with the syndrome myoclonus epilepsy with ragged-red fibres (MERRF) by using fibroblasts and fibroblast-derived cytoplasmic hybrid cells harbouring different percentages of mutated mitochondrial DNA (mtDNA). The activity of cytochrome c oxidase (COX) in patient fibroblasts with 89% mutated mtDNA was decreased to 20% of the control levels. COX exhibited altered kinetics, with a decreased V(max) for both the low-affinity and high-affinity phases; however, the K(m) values were not significantly changed. The substrate-dependent synthesis of ATP was decreased to 50% of the control. Analysis of the mitochondrial membrane potential, DeltaPsi, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow cytometry showed a 80% decrease in DeltaPsi at state 4 and an increased sensitivity of DeltaPsi to an uncoupler in fibroblasts from the patient. The investigation of transmitochondrial cytoplasmic hybrid clones derived from the patient's fibroblasts enabled us to characterize the relationship between heteroplasmy of the MERRF mutation, COX activity and DeltaPsi. Within the range of 87-73% mutated mtDNA, COX activity was decreased to 5-35% and DeltaPsi was decreased to 6-78%. These results demonstrate that the MERRF mutation affects COX activity and DeltaPsi in different proportions with regard to mutation heteroplasmy and indicate that the biochemical manifestation of the MERRF mutation exerts a very steep threshold of DeltaPsi inhibition.

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Selected References

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  1. Arpa J., Campos Y., Gutiérrez-Molina M., Martin-Casanueva M. A., Cruz-Martínez A., Pérez-Conde M. C., López-Pajares R., Morales M. C., Tatay J., Lacasa T. Gene dosage effect in one family with myoclonic epilepsy and ragged-red fibers (MERRF). Acta Neurol Scand. 1997 Aug;96(2):65–71. doi: 10.1111/j.1600-0404.1997.tb00241.x. [DOI] [PubMed] [Google Scholar]
  2. Attardi G., Yoneda M., Chomyn A. Complementation and segregation behavior of disease-causing mitochondrial DNA mutations in cellular model systems. Biochim Biophys Acta. 1995 May 24;1271(1):241–248. doi: 10.1016/0925-4439(95)00034-2. [DOI] [PubMed] [Google Scholar]
  3. Baracca A., Curatola G., Parenti Castelli G., Solaini G. The kinetic and structural changes of the mitochondrial F1-ATPase with temperature. Biochem Biophys Res Commun. 1986 May 14;136(3):891–898. doi: 10.1016/0006-291x(86)90416-x. [DOI] [PubMed] [Google Scholar]
  4. Boulet L., Karpati G., Shoubridge E. A. Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF). Am J Hum Genet. 1992 Dec;51(6):1187–1200. [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Burrin J. M., Paterson J. L., Hall G. M. Simple enzymatic method for plasma branched-chain amino acids. Clin Chim Acta. 1985 Nov 29;153(1):37–42. doi: 10.1016/0009-8981(85)90136-6. [DOI] [PubMed] [Google Scholar]
  7. Chinnery P. F., Howell N., Lightowlers R. N., Turnbull D. M. Molecular pathology of MELAS and MERRF. The relationship between mutation load and clinical phenotypes. Brain. 1997 Oct;120(Pt 10):1713–1721. doi: 10.1093/brain/120.10.1713. [DOI] [PubMed] [Google Scholar]
  8. Chomyn A., Lai S. T., Shakeley R., Bresolin N., Scarlato G., Attardi G. Platelet-mediated transformation of mtDNA-less human cells: analysis of phenotypic variability among clones from normal individuals--and complementation behavior of the tRNALys mutation causing myoclonic epilepsy and ragged red fibers. Am J Hum Genet. 1994 Jun;54(6):966–974. [PMC free article] [PubMed] [Google Scholar]
  9. Chomyn A., Meola G., Bresolin N., Lai S. T., Scarlato G., Attardi G. In vitro genetic transfer of protein synthesis and respiration defects to mitochondrial DNA-less cells with myopathy-patient mitochondria. Mol Cell Biol. 1991 Apr;11(4):2236–2244. doi: 10.1128/mcb.11.4.2236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chomyn A. The myoclonic epilepsy and ragged-red fiber mutation provides new insights into human mitochondrial function and genetics. Am J Hum Genet. 1998 Apr;62(4):745–751. doi: 10.1086/301813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Constantin-Teodosiu D., Cederblad G., Hultman E. A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue. Anal Biochem. 1991 Nov 1;198(2):347–351. doi: 10.1016/0003-2697(91)90437-x. [DOI] [PubMed] [Google Scholar]
  12. Enriquez J. A., Chomyn A., Attardi G. MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination. Nat Genet. 1995 May;10(1):47–55. doi: 10.1038/ng0595-47. [DOI] [PubMed] [Google Scholar]
  13. Fabrizi G. M., Cardaioli E., Grieco G. S., Cavallaro T., Malandrini A., Manneschi L., Dotti M. T., Federico A., Guazzi G. The A to G transition at nt 3243 of the mitochondrial tRNALeu(UUR) may cause an MERRF syndrome. J Neurol Neurosurg Psychiatry. 1996 Jul;61(1):47–51. doi: 10.1136/jnnp.61.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fang W., Huang C. C., Chu N. S., Lee C. C., Chen R. S., Pang C. Y., Shih K. D., Wei Y. H. Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome: report of a Chinese family with mitochondrial DNA point mutation in tRNA(Lys) gene. Muscle Nerve. 1994 Jan;17(1):52–57. doi: 10.1002/mus.880170107. [DOI] [PubMed] [Google Scholar]
  15. Glerum D. M., Yanamura W., Capaldi R. A., Robinson B. H. Characterization of cytochrome-c oxidase mutants in human fibroblasts. FEBS Lett. 1988 Aug 15;236(1):100–104. doi: 10.1016/0014-5793(88)80293-x. [DOI] [PubMed] [Google Scholar]
  16. Hammans S. R., Sweeney M. G., Brockington M., Lennox G. G., Lawton N. F., Kennedy C. R., Morgan-Hughes J. A., Harding A. E. The mitochondrial DNA transfer RNA(Lys)A-->G(8344) mutation and the syndrome of myoclonic epilepsy with ragged red fibres (MERRF). Relationship of clinical phenotype to proportion of mutant mitochondrial DNA. Brain. 1993 Jun;116(Pt 3):617–632. doi: 10.1093/brain/116.3.617. [DOI] [PubMed] [Google Scholar]
  17. Hammans S. R., Sweeney M. G., Brockington M., Morgan-Hughes J. A., Harding A. E. Mitochondrial encephalopathies: molecular genetic diagnosis from blood samples. Lancet. 1991 Jun 1;337(8753):1311–1313. doi: 10.1016/0140-6736(91)92981-7. [DOI] [PubMed] [Google Scholar]
  18. Hayashi J., Ohta S., Kikuchi A., Takemitsu M., Goto Y., Nonaka I. Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10614–10618. doi: 10.1073/pnas.88.23.10614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Houstek J., Kopecký J., Zanotti F., Guerrieri F., Jirillo E., Capozza G., Papa S. Topological and functional characterization of the F0I subunit of the membrane moiety of the mitochondrial H+-ATP synthase. Eur J Biochem. 1988 Apr 5;173(1):1–8. doi: 10.1111/j.1432-1033.1988.tb13959.x. [DOI] [PubMed] [Google Scholar]
  20. Houstek J., Vízek K., Pavelka S., Kopecký J., Krejcová E., Hermanská J., Cermáková M. Type II iodothyronine 5'-deiodinase and uncoupling protein in brown adipose tissue of human newborns. J Clin Endocrinol Metab. 1993 Aug;77(2):382–387. doi: 10.1210/jcem.77.2.8393883. [DOI] [PubMed] [Google Scholar]
  21. James A. M., Wei Y. H., Pang C. Y., Murphy M. P. Altered mitochondrial function in fibroblasts containing MELAS or MERRF mitochondrial DNA mutations. Biochem J. 1996 Sep 1;318(Pt 2):401–407. doi: 10.1042/bj3180401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. King M. P., Attardi G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science. 1989 Oct 27;246(4929):500–503. doi: 10.1126/science.2814477. [DOI] [PubMed] [Google Scholar]
  23. Klement P., Nijtmans L. G., Van den Bogert C., Houstek J. Analysis of oxidative phosphorylation complexes in cultured human fibroblasts and amniocytes by blue-native-electrophoresis using mitoplasts isolated with the help of digitonin. Anal Biochem. 1995 Oct 10;231(1):218–224. doi: 10.1006/abio.1995.1523. [DOI] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Larsson N. G., Tulinius M. H., Holme E., Oldfors A., Andersen O., Wahlström J., Aasly J. Segregation and manifestations of the mtDNA tRNA(Lys) A-->G(8344) mutation of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome. Am J Hum Genet. 1992 Dec;51(6):1201–1212. [PMC free article] [PubMed] [Google Scholar]
  26. Lombes A., Nakase H., Tritschler H. J., Kadenbach B., Bonilla E., DeVivo D. C., Schon E. A., DiMauro S. Biochemical and molecular analysis of cytochrome c oxidase deficiency in Leigh's syndrome. Neurology. 1991 Apr;41(4):491–498. doi: 10.1212/wnl.41.4.491. [DOI] [PubMed] [Google Scholar]
  27. Masucci J. P., Davidson M., Koga Y., Schon E. A., King M. P. In vitro analysis of mutations causing myoclonus epilepsy with ragged-red fibers in the mitochondrial tRNA(Lys)gene: two genotypes produce similar phenotypes. Mol Cell Biol. 1995 May;15(5):2872–2881. doi: 10.1128/mcb.15.5.2872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  29. Nijtmans L. G., Barth P. G., Lincke C. R., Van Galen M. J., Zwart R., Klement P., Bolhuis P. A., Ruitenbeek W., Wanders R. J., Van den Bogert C. Altered kinetics of cytochrome c oxidase in a patient with severe mitochondrial encephalomyopathy. Biochim Biophys Acta. 1995 Apr 24;1270(2-3):193–201. doi: 10.1016/0925-4439(95)00044-5. [DOI] [PubMed] [Google Scholar]
  30. Nijtmans L. G., Klement P., Houstek J., van den Bogert C. Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases. Biochim Biophys Acta. 1995 Dec 12;1272(3):190–198. doi: 10.1016/0925-4439(95)00087-9. [DOI] [PubMed] [Google Scholar]
  31. Noer A. S., Sudoyo H., Lertrit P., Thyagarajan D., Utthanaphol P., Kapsa R., Byrne E., Marzuki S. A tRNA(Lys) mutation in the mtDNA is the causal genetic lesion underlying myoclonic epilepsy and ragged-red fiber (MERRF) syndrome. Am J Hum Genet. 1991 Oct;49(4):715–722. [PMC free article] [PubMed] [Google Scholar]
  32. Ozawa M., Nishino I., Horai S., Nonaka I., Goto Y. I. Myoclonus epilepsy associated with ragged-red fibers: a G-to-A mutation at nucleotide pair 8363 in mitochondrial tRNA(Lys) in two families. Muscle Nerve. 1997 Mar;20(3):271–278. doi: 10.1002/(SICI)1097-4598(199703)20:3<271::AID-MUS2>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  33. Piccolo G., Focher F., Verri A., Spadari S., Banfi P., Gerosa E., Mazzarello P. Myoclonus epilepsy and ragged-red fibers: blood mitochondrial DNA heteroplasmy in affected and asymptomatic members of a family. Acta Neurol Scand. 1993 Dec;88(6):406–409. doi: 10.1111/j.1600-0404.1993.tb05368.x. [DOI] [PubMed] [Google Scholar]
  34. Schon E. A., Bonilla E., DiMauro S. Mitochondrial DNA mutations and pathogenesis. J Bioenerg Biomembr. 1997 Apr;29(2):131–149. doi: 10.1023/a:1022685929755. [DOI] [PubMed] [Google Scholar]
  35. Schägger H., von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem. 1991 Dec;199(2):223–231. doi: 10.1016/0003-2697(91)90094-a. [DOI] [PubMed] [Google Scholar]
  36. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  37. Seibel P., Degoul F., Romero N., Marsac C., Kadenbach B. Identification of point mutations by mispairing PCR as exemplified in MERRF disease. Biochem Biophys Res Commun. 1990 Dec 14;173(2):561–565. doi: 10.1016/s0006-291x(05)80071-3. [DOI] [PubMed] [Google Scholar]
  38. Shih K. D., Yen T. C., Pang C. Y., Wei Y. H. Mitochondrial DNA mutation in a Chinese family with myoclonic epilepsy and ragged-red fiber disease. Biochem Biophys Res Commun. 1991 Feb 14;174(3):1109–1116. doi: 10.1016/0006-291x(91)91535-k. [DOI] [PubMed] [Google Scholar]
  39. Shoffner J. M., Lott M. T., Lezza A. M., Seibel P., Ballinger S. W., Wallace D. C. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation. Cell. 1990 Jun 15;61(6):931–937. doi: 10.1016/0092-8674(90)90059-n. [DOI] [PubMed] [Google Scholar]
  40. Silvestri G., Moraes C. T., Shanske S., Oh S. J., DiMauro S. A new mtDNA mutation in the tRNA(Lys) gene associated with myoclonic epilepsy and ragged-red fibers (MERRF). Am J Hum Genet. 1992 Dec;51(6):1213–1217. [PMC free article] [PubMed] [Google Scholar]
  41. Spelbrink J. N., Van Oost B. A., Van den Bogert C. The relationship between mitochondrial genotype and mitochondrial phenotype in lymphoblasts with a heteroplasmic mtDNA deletion. Hum Mol Genet. 1994 Nov;3(11):1989–1997. doi: 10.1093/hmg/3.11.1989. [DOI] [PubMed] [Google Scholar]
  42. Sperl W., Ruitenbeek W., Kerkhof C. M., Sengers R. C., Trijbels J. M., Guggenbichler J. P., Janssen A. J., Bakkeren J. A. Deficiency of the alpha and beta subunits of pyruvate dehydrogenase in a patient with lactic acidosis and unexpected sudden death. Eur J Pediatr. 1990 Apr;149(7):487–492. doi: 10.1007/BF01959401. [DOI] [PubMed] [Google Scholar]
  43. Tiranti V., Chariot P., Carella F., Toscano A., Soliveri P., Girlanda P., Carrara F., Fratta G. M., Reid F. M., Mariotti C. Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNASer(UCN) gene. Hum Mol Genet. 1995 Aug;4(8):1421–1427. doi: 10.1093/hmg/4.8.1421. [DOI] [PubMed] [Google Scholar]
  44. Tiranti V., Munaro M., Sandonà D., Lamantea E., Rimoldi M., DiDonato S., Bisson R., Zeviani M. Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho degrees transformants. Hum Mol Genet. 1995 Nov;4(11):2017–2023. doi: 10.1093/hmg/4.11.2017. [DOI] [PubMed] [Google Scholar]
  45. Villani G., Attardi G. In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1166–1171. doi: 10.1073/pnas.94.4.1166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wanders R. J., Van Roermund C. W. Studies on phytanic acid alpha-oxidation in rat liver and cultured human skin fibroblasts. Biochim Biophys Acta. 1993 Apr 23;1167(3):345–350. doi: 10.1016/0005-2760(93)90239-6. [DOI] [PubMed] [Google Scholar]
  47. Yoneda M., Miyatake T., Attardi G. Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles. Mol Cell Biol. 1994 Apr;14(4):2699–2712. doi: 10.1128/mcb.14.4.2699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zeviani M., Amati P., Bresolin N., Antozzi C., Piccolo G., Toscano A., DiDonato S. Rapid detection of the A----G(8344) mutation of mtDNA in Italian families with myoclonus epilepsy and ragged-red fibers (MERRF). Am J Hum Genet. 1991 Feb;48(2):203–211. [PMC free article] [PubMed] [Google Scholar]
  49. Zeviani M., Muntoni F., Savarese N., Serra G., Tiranti V., Carrara F., Mariotti C., DiDonato S. A MERRF/MELAS overlap syndrome associated with a new point mutation in the mitochondrial DNA tRNA(Lys) gene. Eur J Hum Genet. 1993;1(1):80–87. doi: 10.1159/000472390. [DOI] [PubMed] [Google Scholar]

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