Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1989 May;83(5):1605–1613. doi: 10.1172/JCI114058

Molecular cloning and nucleotide sequence of complementary DNAs encoding human short chain acyl-coenzyme A dehydrogenase and the study of the molecular basis of human short chain acyl-coenzyme A dehydrogenase deficiency.

E Naito 1, H Ozasa 1, Y Ikeda 1, K Tanaka 1
PMCID: PMC303867  PMID: 2565344

Abstract

Complementary DNAs encoding the precursor of human placental short chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) (EC 1.3.99.2) were cloned and sequenced. The cDNA inserts in these clones were 1,852 bases in length combined, and encoded the entire 412-amino acid precursor SCAD (mol wt 44,303). This sequence included the 24-amino acid leader peptide moiety (mol wt 2,576) and 388 amino acids corresponding to the mature protein (mol wt 41,727). The comparison of SCAD and medium chain acyl-CoA dehydrogenase sequences revealed a high degree of homology, suggesting that these enzymes evolved from a common ancestral gene and belong to a gene family. We also studied mutant human SCAD in cultured skin fibroblasts from three patients with hereditary SCAD deficiency. Labeling fibroblast cultures with [35S]-methionine followed by immunoprecipitation with anti-SCAD antibody revealed that a normal size variant SCAD protein was synthesized. In all of the three SCAD-deficient cell lines, the size of variant SCAD mRNA as determined by Northern blotting using one of the normal SCAD cDNA as a probe was also normal, and no difference was observed on Southern blots in the restriction patterns of mutant genomic DNA using EcoRI, TaqI, HincII, and BamHI. These results suggest that the defects in SCAD in these cell lines are caused by a point mutation.

Full text

PDF
1605

Images in this article

1608Image
on p.1608
1608Image
on p.1608
1608Image
on p.1608
1610Image
on p.1610
1611Image
on p.1611
1611Image
on p.1611
1612Image
on p.1612
1612Image
on p.1612
1612Image
on p.1612

Selected References

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

  1. Amendt B. A., Greene C., Sweetman L., Cloherty J., Shih V., Moon A., Teel L., Rhead W. J. Short-chain acyl-coenzyme A dehydrogenase deficiency. Clinical and biochemical studies in two patients. J Clin Invest. 1987 May;79(5):1303–1309. doi: 10.1172/JCI112953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Coates P. M., Hale D. E., Finocchiaro G., Tanaka K., Winter S. C. Genetic deficiency of short-chain acyl-coenzyme A dehydrogenase in cultured fibroblasts from a patient with muscle carnitine deficiency and severe skeletal muscle weakness. J Clin Invest. 1988 Jan;81(1):171–175. doi: 10.1172/JCI113290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Duran M., Walther F. J., Bruinvis L., Wadman S. K. The urinary excretion of ethylmalonic acid: what level requires further attention? Biochem Med. 1983 Apr;29(2):171–175. doi: 10.1016/0006-2944(83)90037-6. [DOI] [PubMed] [Google Scholar]
  4. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  5. Finocchiaro G., Ito M., Tanaka K. Purification and properties of short chain acyl-CoA, medium chain acyl-CoA, and isovaleryl-CoA dehydrogenases from human liver. J Biol Chem. 1987 Jun 15;262(17):7982–7989. [PubMed] [Google Scholar]
  6. Hay R., Böhni P., Gasser S. How mitochondria import proteins. Biochim Biophys Acta. 1984 Jan 27;779(1):65–87. doi: 10.1016/0304-4157(84)90004-2. [DOI] [PubMed] [Google Scholar]
  7. Ikeda Y., Hine D. G., Okamura-Ikeda K., Tanaka K. Mechanism of action of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases. Direct evidence for carbanion formation as an intermediate step using enzyme-catalyzed C-2 proton/deuteron exchange in the absence of C-3 exchange. J Biol Chem. 1985 Jan 25;260(2):1326–1337. [PubMed] [Google Scholar]
  8. Ikeda Y., Keese S. M., Fenton W. A., Tanaka K. Biosynthesis of four rat liver mitochondrial acyl-CoA dehydrogenases: in vitro synthesis, import into mitochondria, and processing of their precursors in a cell-free system and in cultured cells. Arch Biochem Biophys. 1987 Feb 1;252(2):662–674. doi: 10.1016/0003-9861(87)90072-5. [DOI] [PubMed] [Google Scholar]
  9. Ikeda Y., Keese S. M., Tanaka K. Molecular heterogeneity of variant isovaleryl-CoA dehydrogenase from cultured isovaleric acidemia fibroblasts. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7081–7085. doi: 10.1073/pnas.82.20.7081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ikeda Y., Okamura-Ikeda K., Tanaka K. Purification and characterization of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria. Isolation of the holo- and apoenzymes and conversion of the apoenzyme to the holoenzyme. J Biol Chem. 1985 Jan 25;260(2):1311–1325. [PubMed] [Google Scholar]
  11. Ikeda Y., Tanaka K. Immunoprecipitation and electrophoretic analysis of four human acyl-CoA dehydrogenases and electron transfer flavoprotein using antibodies raised against the corresponding rat enzymes. Biochem Med Metab Biol. 1987 Jun;37(3):329–334. doi: 10.1016/0885-4505(87)90044-2. [DOI] [PubMed] [Google Scholar]
  12. Ikeda Y., Tanaka K. Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria. J Biol Chem. 1983 Aug 10;258(15):9477–9487. [PubMed] [Google Scholar]
  13. Ikeda Y., Tanaka K. Purification and characterization of isovaleryl coenzyme A dehydrogenase from rat liver mitochondria. J Biol Chem. 1983 Jan 25;258(2):1077–1085. [PubMed] [Google Scholar]
  14. Kelly D. P., Kim J. J., Billadello J. J., Hainline B. E., Chu T. W., Strauss A. W. Nucleotide sequence of medium-chain acyl-CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4068–4072. doi: 10.1073/pnas.84.12.4068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Matsubara Y., Kraus J. P., Ozasa H., Glassberg R., Finocchiaro G., Ikeda Y., Mole J., Rosenberg L. E., Tanaka K. Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase. J Biol Chem. 1987 Jul 25;262(21):10104–10108. [PubMed] [Google Scholar]
  17. Matsubara Y., Kraus J. P., Yang-Feng T. L., Francke U., Rosenberg L. E., Tanaka K. Molecular cloning of cDNAs encoding rat and human medium-chain acyl-CoA dehydrogenase and assignment of the gene to human chromosome 1. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6543–6547. doi: 10.1073/pnas.83.17.6543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nevins J. R. The pathway of eukaryotic mRNA formation. Annu Rev Biochem. 1983;52:441–466. doi: 10.1146/annurev.bi.52.070183.002301. [DOI] [PubMed] [Google Scholar]
  19. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tanaka K., Ikeda Y., Matsubara Y., Hyman D. B. Molecular basis of isovaleric acidemia and medium-chain acyl-CoA dehydrogenase deficiency. Enzyme. 1987;38(1-4):91–107. doi: 10.1159/000469195. [DOI] [PubMed] [Google Scholar]
  22. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES