Cloning of a cDNA encoding bovine mitochondrial NADP(+)-specific isocitrate dehydrogenase and structural comparison with its isoenzymes from different species.
Mitochondrial NADP(+)-specific isocitrate dehydrogenase (IDP) was co-purified with the pyruvate dehydrogenase complex from bovine kidney mitochondria. The determination of its N-terminal 16-amino-acid sequence revealed that it is highly similar to the IDP from yeast. A cDNA clone (1.8 kb long) encoding this protein was isolated from a bovine kidney lambda gt11 cDNA library using a synthetic oligodeoxynucleotide. The deduced protein sequence of this cDNA clone rendered a precursor protein of 452 amino-acid residues (50,830 Da) and a mature protein of 413 amino-acid residues (46,519 Da). It is 100% identical to the internal tryptic peptide sequences of the autologous form from pig heart and 62% similar to that from yeast. However, it shares little similarity with the mitochondrial NAD(+)-specific isoenzyme from yeast. Structural analyses of the deduced proteins of IDP isoenzymes from different species indicated that similarity exists in certain regions, which may represent the common domains for the active sites or coenzyme-binding sites. In Northern-blot analysis, one species of mRNA (about 2.2 kb for both bovine and human) was hybridized with a 32P-labelled cDNA probe. Southern-blot analysis of genomic DNAs verified simple patterns of hybridization with this cDNA. These results strongly indicate that the mitochondrial IDP may be derived from a single gene family which does not appear to be closely related to that of the NAD(+)-specific isoenzyme.
Huh
T L
TL
Department of Genetic Engineering, College of Natural Sciences, Kyungpook National University, Taegu, Korea.
Ryu
J H
JH
Huh
J W
JW
Sung
H C
HC
Oh
I U
IU
Song
B J
BJ
Veech
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004247:DNA; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011768:Pyruvate Dehydrogenase Complex; D012333:RNA, Messenger; D009249:NADP; D004247:DNA; D007521:Isocitrate Dehydrogenase