Human mitochondrial NAD-specific isocitrate dehydrogenase 3 and subunits: Cloning, tissue specific expression and functional analyses of the recombinant proteins

T. L. Huh, Y. O. Kim, H. J. Ko, S. H. Kim, Hae Chul Park, L. J. Lee, B. J. Song

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Mammalian mitochondrial NAD+-specific isocitrate dehydrogenase is composed of three nonidentical subunits, designated IDHcq IDH3 and IDHy. We report the cloning and characterization of full-length eDNA clones for the human IDH[3 and IDtty subunits. The deduced protein sequences from human IDH[3 and t' rendered mature proteins of 351 (Mr 38,794) and 354 (M, 38,814) amino acids, respectively. The human IDH!3 and y subunits share 53% amino acid similarity while they are less similar (44%) to the a subunit. Northern blot analyses revealed tissue-specific expressions of IDH mRNA transcripts. The IDH!3 and IDHy subunits were expressed in E. coil in various combinations with the previously cloned IDH c subunit. The recombinant IDHct[3, cy and oq3y co-produced in bacteria exhibited IDH enzyme activity, whereas ct, [3 and y alone and fly were inactive. The Km values of IDH[3, my and cti3y were 2.71, 7.31 and 0.76 mM respectively. Addition of Ca2+(20 gM) decreased the Km values of 1DHct[3, cq' and etDy were decreased to 2.4, 9.1, and 3 - fold, respectively while a 4fold increase in Vmax value was observed with the IDHcty protein. These results indicate that the cx subunit is essential for the catalytic activity and that each of the [3 and y subunits is likely to play a different regulatory role (with a possibility of the ,; subunit acting as the primary Ca:+ binding site). The enzymatically active recombinant IDHcq3y protein was partially purified on a gel filtration column and eluted as a single peak (316 kDa), suggesting that the catalytically active IDH enzyme exists as an octamer.

Original languageEnglish
JournalFASEB Journal
Volume11
Issue number9
Publication statusPublished - 1997 Dec 1
Externally publishedYes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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