Bovine NAD(+)-dependent isocitrate dehydrogenase: Alternative splicing andtissue-dependent expression of subunit 1

NAD(+)-dependent isocitrate dehydrogenase (IDH), a key regulatory enzyme in the Krebs cycle, is a multi-tetrameric enzyme. At least three of the subun its in the core tetramer of mammals are unique gene products. Subunits 1/be ta and 2/gamma are considered to be regulatory, while subunits 3,4/alpha, c omprising 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 I-B). Differences in the two were confin ed to the 3'-region, suggesting alternative splicing. Screening of brain, k idney, and liver RNA showed the presence of IDH 1-A and 1-B and a third maj or 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 s plice junctions between exon W, exon X, and exon Y. IDH 1-C resulted from s plicing of exon W to exon X and subsequent retention of intron x. The 2 pro teins predicted from these 3 mRNAs are identical over their first 357 resid ues. Protein IDH 1-A, resulting from a termination codon within exon Y, con tains an additional 26 residues. Proteins IDH 1-B and 1-C derive from a com mon termination codon within exon X and contain an additional 28 residues. The two C-terminal regions differ notably in the number and nature of charg ed 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 alternat ive splicing in subunit 3,4/alpha. The results of the present study, togeth er with recent work on the 2/gamma subunit [Brenner,V., Nyakatura, G., Rose nthal, A., and Platter, M. (1998) Genomics 44, 8], indicate that the regula tory subunits of the enzyme, but not the catalytic, possess alternatively s pliced forms varying in C-terminal properties with tissue-specific expressi on. The finding is suggestive of a mechanism for modulation of allosteric r egulation tailored to the needs of different tissues.