Summary
The abundance and phosphorylation state of the polypeptide constitutents of the human branched-chain 2-oxo acid dehydrogenase complex were examined in mitochondria from normal and maple syrup urine disease (MSUD) fibroblasts. In normal fibroblast mitochondria two forms of the E1α subunit were observed: non-phosphorylated (E1α) and phosphorylated (E1α−P). About 40–50% of E1α was present as E1α−P. The ability to quantitate the two forms of E1α permitted examination of the association between decreased capacity to oxidize branched-chain 2-oxo acids and the phosphorylation state of E1α.
Changes in phosphorylation state of E1α were observed in MSUD fibroblasts as compared to control cells. Of particular interest was the absence of E1α−P in an MSUD fibroblast line which lacked the dihydrolipoyl acyltransferase (E2) subunit of the dehydrogenase complex. In two MSUD cell lines deficient in E1α, the abundance of E1α−P appeared to be preferentially reduced. A fourth MSUD cell line contained normal quantities of E3, E2 and both forms of the E1α polypeptide. Our results indicate that alterations in the abundance of dehydrogenase complex polypeptides in MSUD fibroblasts may influence the phosphorylation state of the E1α polypeptide. They demonstrate the potential for examining simultaneously mutations which affect both the catalytic and regulatory components of the dehydrogenase complex.
Similar content being viewed by others
References
Chuang, D. T., Niu, W-L. and Cox, R. P. Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical maple syrup urine disease subjects.Biochem. J. 200 (1981) 59–67
Chuang, D. T., Ku, L. S., Kerr, D. S. and Cox, R. P. Detection of heterozygotes in maple syrup urine disease: Measurements of branched-chain α-ketoacid dehydrogenase and its components in cell cultures.Am. J. Hum. Genet. 34 (1982) 416–424
Cook, K. G., Bradford, A. P. and Yeaman, S. J. Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex.Biochem. J. 225 (1985) 731–735
Dancis, J., Hutzler, J. and Cox, R. P. Maple syrup urine disease: Branched-chain keto acid decarboxylation in fibroblasts as measured with amino acids and keto acids.Am. J. Hum. Genet. 29 (1977) 272–279
Danner, D. J. and Elsas, L. J. Disorders of branched chain amino acid and keto acid metabolism. In Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D. (eds.),The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1989, pp. 671–692
Danner, D. J. and Priest, J. H. Branched-chain ketoacid dehydrogenase activity and growth of normal and mutant human fibroblasts: The effect of branched-chain amino acid concentration in culture medium.Biochem. Genet. 21 (1983) 895–905
Danner, D. J., Armstrong, N., Heffelfinger, S. C., Sewell, E. T., Priest, J. H. and Elsas, L.J. Absence of branched chain acyltransferase as a cause of maple syrup urine disease.J. Clin. Invest. 75 (1985) 858–860
Eisenstein, R. S., Miller, R. H., Hoganson, G. and Harper, A. E. Phylogenetic comparisons of the branched-chain α-keto acid dehydrogenase complex.Comp. Biochem. Physiol. (1990) (in press)
Elsas, L. J. Perinatal diagnosis of the inborn errors of metabolism.J. Med. Assoc. Georgia 60 (1971) 308–311
Elsas, L. J., Pask, B. A., Wheeler, F. B., Perl, D. P. and Trusler, S. Classical maple syrup urine disease: cofactor resistance.Metabolism 21 (1972) 929–944
Fensom, A. H., Benson, P. F. and Baker, J. E. A rapid method for assay of branched-chain keto acid decarboxylation in cultured cells and its application to prenatal diagnosis of maple syrup urine disease.Clin. Chim. Acta 87 (1978) 169–174
Fischer, C. W., Chuang, J. L., Griffin, T. A., Lau, K. S., Cox, R. P. and Chuang, D. T. Molecular phenotypes in cultured maple syrup urine disease cells.J. Biol. Chem. 264 (1989) 3448–3453
Harris, R. A., Paxton, R. and DePaoli-Roach, A. A. Inhibition of branched chainα-ketoacid dehydrogenase kinase activity byα-chloroisocaproate.J. Biol. Chem. 257 (1982) 13915–13918
Indo, Y., Kitano, A., Endo, F., Akaboshi, I. and Matsuda, I. Altered kinetic properties of the branched-chainα-keto acid dehydrogenase complex due to mutation of theβ-subunit of the branched-chainα-ketoacid dehydrogenase (E1) component in lymphoblastoid cells derived from patients with maple syrup urine disease.J. Clin. Invest. 80 (1987) 63–70
Kuntz, M. J., Paxton, R., Shimomura, Y. U., Goodwin, G. W. and Harris, R. A. Phosphorylation affects the mobility of the E1α-subunit of branched-chain 2-oxo acid dehydrogenase on sodium dodecyl sulfate/polyacrylamide-gel electrophoresis.Biochem. Soc. Trans. 14 (1986) 1077–1078
Lau, K. S., Griffin, T. A., Hu, C-W. C. and Chuang, D. T. Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chainα-keto acid dehydrogenase complex: Molecular cloning of human and bovine transacylase (E2) cDNAs.Biochemistry 27 (1988) 1972–1981
Miller, R. H., Eisenstein, R. S. and Harper, A. E. Tissue specific alterations in the E1α subunit of branched-chain ketoacid dehydrogenase in rat.Fed. Proc. 45 (1986) 360
Miller, R. H., Eisenstein, R. S. and Harper, A. E. Effects of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat.J. Biol. Chem. 263 (1988) 3454–3461
Millis, R. J. T. and Pious, D. A. Oxidative phosphorylation in mitochondria isolated from human fibroblasts.Biochim. Biophys. Acta 292 (1973) 73–77
Paxton, R. and Harris, R. A. Isolation of rabbit liver branched chainα-ketoacid dehydrogenase and regulation by phosphorylation.J. Biol. Chem. 257 (1982) 14433–14439
Paxton, R. and Harris, R. A. Regulation of branched-chainα-ketoacid dehydrogenase kinase.Arch. Biochem. Biophys. 231 (1984) 48–57
Pettit, F. H., Yeaman, S. J. and Reed, L. J. Purification and characterization of branched chainα-keto acid dehydrogenase complex of bovine kidney.Proc. Natl. Acad. Sci. USA 75 (1978) 4881–4885
Reed, L. J. and Hacker, M. L. Structure-function relationships in dihydrolipoamide acyltransferases.J. Biol. Chem. 265 (1990) 8971–8974
Toshima, K., Kuroda, Y., Yokota, I., Naito, E., Ito, M., Watanabe, T., Takeda, E. and Miyao, M. Activation of branched-chainα-ketoacid dehydrogenase complex byα-chloroisocaproate in normal and enzyme-deficient fibroblasts.Clin. Chim. Acta 147 (1985) 103–108
Yang, M. M. and Youvan, D. C. Application of imaging spectroscopy in molecular biology. I. Screening photosynthetic bacteria.Biotechnology 6 (1988) 939–942
Yeaman, S. J. The 2-oxo acid dehydrogenase complexes: Recent advances.Biochem. J. 257 (1989) 625–632
Zhang, B., Edenberg, H. J., Crabb, D. W. and Harris, R. A. Evidence for both a regulatory mutation and structural mutation in a family with maple syrup urine disease.J. Clin. Invest. 83 (1989) 1425–1429
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Eisenstein, R.S., Hoganson, G., Miller, R.H. et al. Altered phosphorylation state of branched-chain 2-Oxo acid dehydrogenase in a branched-chain acyltransferase deficient human fibroblast cell line. J Inherit Metab Dis 14, 37–44 (1991). https://doi.org/10.1007/BF01804386
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01804386