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.
|Publication status||Published - 1997|
ASJC Scopus subject areas
- Molecular Biology