Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Dec 1;320(Pt 2):451–457. doi: 10.1042/bj3200451

Immunochemical detection of CoA-modified mitochondrial matrix proteins.

W Huth 1, C Pauli 1, U Möller 1
PMCID: PMC1217951  PMID: 8973552

Abstract

An in vitro assay mixture consisting of mitochondrial matrix proteins from rat liver and CoA resulted in the formation of CoA-modified proteins. CoA-modified proteins were demonstrated by detection of CoA. CoA was released from the proteins by dithioerythritol treatment under denaturing conditions and was identified by (a) its retention time on HPLC, (b) its absorption spectrum and (c) its activity in a CoA-specific assay. This CoA represents protein-bound CoA and, in addition, protein-bound palmitoyl-CoA when MgATP was also present in the in vitro assay. The detection of immunoreactive proteins using mono-specific anti-CoA antibodies exclusively identifies CoA-modified proteins. The specificity of these antibodies can be used to identify both endogenously occurring CoA-modified proteins and proteins that have been modified in the in vitro assay. An intact thiol group of CoA is an essential precondition for the modification to occur.

Full Text

The Full Text of this article is available as a PDF (319.5 KB).

Selected References

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

  1. Fukao T., Kamijo K., Osumi T., Fujiki Y., Yamaguchi S., Orii T., Hashimoto T. Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat mitochondrial acetoacetyl-CoA thiolase. J Biochem. 1989 Aug;106(2):197–204. doi: 10.1093/oxfordjournals.jbchem.a122832. [DOI] [PubMed] [Google Scholar]
  2. Helmerhorst E., Stokes G. B. Microcentrifuge desalting: a rapid, quantitative method for desalting small amounts of protein. Anal Biochem. 1980 May 1;104(1):130–135. doi: 10.1016/0003-2697(80)90287-0. [DOI] [PubMed] [Google Scholar]
  3. Huth W., Alves F. Mitochondrial acetyl-CoA acetyltransferase in various organs from rat: form patterns and coenzyme-A-mediated modification. Biochim Biophys Acta. 1985 Aug 23;830(3):274–281. doi: 10.1016/0167-4838(85)90283-3. [DOI] [PubMed] [Google Scholar]
  4. Huth W., Arvand M., Möller U. Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins. Eur J Biochem. 1988 Mar 15;172(3):607–614. doi: 10.1111/j.1432-1033.1988.tb13932.x. [DOI] [PubMed] [Google Scholar]
  5. Huth W. The charge heterogeneity of the mitochondrial acetyl-CoA acetyltransferase from rat liver. Eur J Biochem. 1981 Dec;120(3):557–562. doi: 10.1111/j.1432-1033.1981.tb05736.x. [DOI] [PubMed] [Google Scholar]
  6. Huth W., Worm-Breitgoff C., Möller U., Wunderlich I. Evidence for an in vivo modification of mitochondrial proteins by coenzyme A. Biochim Biophys Acta. 1991 Mar 8;1077(1):1–10. doi: 10.1016/0167-4838(91)90518-5. [DOI] [PubMed] [Google Scholar]
  7. Jonas R., Huth W. Acetyl-CoA acetyltransferase from bovine liver mitochondria. Molecular properties of multiple forms. Biochim Biophys Acta. 1978 Dec 8;527(2):379–390. doi: 10.1016/0005-2744(78)90352-2. [DOI] [PubMed] [Google Scholar]
  8. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Messner D. J., Griffiths G., Kornfeld S. Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors. J Cell Biol. 1989 Jun;108(6):2149–2162. doi: 10.1083/jcb.108.6.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Middleton B. The existence of ketoacyl-CoA thiolases of differing properties and intracellular localization in ox liver. Biochem Biophys Res Commun. 1972 Jan 31;46(2):508–515. doi: 10.1016/s0006-291x(72)80168-2. [DOI] [PubMed] [Google Scholar]
  12. Middleton B. The oxoacyl-coenzyme A thiolases of animal tissues. Biochem J. 1973 Apr;132(4):717–730. doi: 10.1042/bj1320717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Odaka M., Kiribuchi K., Allison W. S., Yoshida M. In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond. FEBS Lett. 1993 Dec 27;336(2):231–235. doi: 10.1016/0014-5793(93)80809-9. [DOI] [PubMed] [Google Scholar]
  14. Quandt L., Huth W. Modulation of rat-liver mitochondrial acetyl-CoA acetyltransferase activity by a reversible chemical modification with coenzyme A. Biochim Biophys Acta. 1984 Jan 31;784(2-3):168–176. doi: 10.1016/0167-4838(84)90124-9. [DOI] [PubMed] [Google Scholar]
  15. Quandt L., Huth W. On the mechanism of the chemical modification of the mitochondrial acetyl-CoA acetyltransferase by coenzyme A. Biochim Biophys Acta. 1985 May 20;829(1):103–108. doi: 10.1016/0167-4838(85)90073-1. [DOI] [PubMed] [Google Scholar]
  16. Schnaitman C., Greenawalt J. W. Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell Biol. 1968 Jul;38(1):158–175. doi: 10.1083/jcb.38.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schwerdt G., Huth W. Turnover and transformation of mitochondrial acetyl-CoA acetyltransferase into CoA-modified forms. Biochem J. 1993 Jun 15;292(Pt 3):915–919. doi: 10.1042/bj2920915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sottocasa G. L., Kuylenstierna B., Ernster L., Bergstrand A. An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol. 1967 Feb;32(2):415–438. doi: 10.1083/jcb.32.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stadtman E. R. Covalent modification reactions are marking steps in protein turnover. Biochemistry. 1990 Jul 10;29(27):6323–6331. doi: 10.1021/bi00479a001. [DOI] [PubMed] [Google Scholar]
  20. Thorneley R. N., Abell C., Ashby G. A., Drummond M. H., Eady R. R., Huff S., Macdonald C. J., Shneier A. Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by 31P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation. Biochemistry. 1992 Feb 4;31(4):1216–1224. doi: 10.1021/bi00119a035. [DOI] [PubMed] [Google Scholar]
  21. von Figura K. Human alpha-N-acetylglucosaminidase. 1. Purification and properties. Eur J Biochem. 1977 Nov 1;80(2):523–533. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES