Bovine NAD+-dependent isocitrate dehydrogenase: alternative splicing and tissue-dependent expression of subunit 1

Biochemistry. 2000 Feb 22;39(7):1807-16. doi: 10.1021/bi991691i.

Abstract

NAD+-dependent isocitrate dehydrogenase (IDH), a key regulatory enzyme in the Krebs cycle, is a multi-tetrameric enzyme. At least three of the subunits in the core tetramer of mammals are unique gene products. Subunits 1/beta and 2/gamma are considered to be regulatory, while subunits 3,4/alpha, comprising half the tetramer, are catalytic. The full sequence was obtained for the major subunit 1 cDNA in bovine heart, IDH 1-A. A second cDNA, rare in heart, was also identified (IDH 1-B). Differences in the two were confined to the 3'-region, suggesting alternative splicing. Screening of brain, kidney, and liver RNA showed the presence of IDH 1-A and 1-B and a third major species, IDH 1-C. Amplification of bovine genomic DNA by PCR across the regions of difference produced a single product. Comparison of the genomic and mRNA sequences showed that IDH 1-A resulted from splicing of exon W to exon Y, eliminating intron w, exon X, and intron x. IDH 1-B was formed by splice junctions between exon W, exon X, and exon Y. IDH 1-C resulted from splicing of exon W to exon X and subsequent retention of intron x. The 2 proteins predicted from these 3 mRNAs are identical over their first 357 residues. Protein IDH 1-A, resulting from a termination codon within exon Y, contains an additional 26 residues. Proteins IDH 1-B and 1-C derive from a common termination codon within exon X and contain an additional 28 residues. The two C-terminal regions differ notably in the number and nature of charged residues, resulting in proteins with a charge difference of 3.2 at pH 7.0. Subunit 1 sequences previously reported from other species grouped with one or the other of the bovine proteins. No evidence was found for alternative splicing in subunit 3,4/alpha. The results of the present study, together with recent work on the 2/gamma subunit [Brenner,V., Nyakatura, G., Rosenthal, A., and Platzer, M. (1998) Genomics 44, 8], indicate that the regulatory subunits of the enzyme, but not the catalytic, possess alternatively spliced forms varying in C-terminal properties with tissue-specific expression. The finding is suggestive of a mechanism for modulation of allosteric regulation tailored to the needs of different tissues.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Brain / metabolism
  • Cattle
  • Gene Expression Regulation
  • Humans
  • Isocitrate Dehydrogenase / biosynthesis*
  • Isocitrate Dehydrogenase / genetics*
  • Kidney / metabolism
  • Liver / metabolism
  • Macaca fascicularis
  • Molecular Sequence Data
  • Myocardium / metabolism
  • NAD / metabolism*
  • Organ Specificity / genetics
  • RNA, Messenger / biosynthesis
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA

Substances

  • RNA, Messenger
  • NAD
  • Isocitrate Dehydrogenase

Associated data

  • GENBANK/AF090321
  • GENBANK/AF090322
  • GENBANK/AF090323
  • GENBANK/AF090324
  • GENBANK/AL050094