Identification and functional characterization of a novel, tissue-specificNAD(+)-dependent isocitrate dehydrogenase beta subunit isoform

To understand the interactions and functional role of each of the three mit ochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH) subunits (alpha, beta, and gamma), we have characterized human cDNAs encoding two beta isof orms (beta(1) and beta(2)) and the gamma subunit. Analysis of deduced amino acid sequences revealed that beta(1) and beta(2) encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The gamma cDNA encodes 354 amino acids and is almost identi cal to monkey IDH gamma. Northern analyses revealed that the smaller beta(2 ) transcript (1.3 kilobases) is primarily expressed in heart and skeletal m uscle, whereas the larger beta(1) mRNA (1.6 kilobases) is prevalent in nonm uscle tissues. Sequence analysis of the IDH beta gene indicates that the di fference in the C-terminal 28 amino acids between beta(1) and beta(2) prote ins results from alternative splicing of a single transcript. Among the var ious combinations of human IDH subunits co-expressed in bacteria, alpha bet a gamma, alpha beta, and alpha gamma combinations exhibited significant amo unts of IDH activity, whereas subunits produced alone and beta gamma showed no detectable activity. These data suggest that the alpha is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of beta(1) with beta(2) in the co-expression system lowered the pH optimum for IDH activity from 8.0 to 7. 6. This difference in optimal pH was analogous to what was observed in mous e kidney and brain (beta(1) prevalent; optimal pH 8.0) versus heart (beta(2 ) prevalent; pH 7.6) mitochondria, Experiments with a specially designed sp licing reporter constrdct stably transfected into HT1080 cells indicate tha t acidic conditions favor a splicing pattern responsible for the muscle- an d heart-specific beta(2) isoform. Taken together, these data indicate a reg ulatory role of IDH beta isoforms in determining the pH optimum for IDH act ivity through the tissue-specific alternative splicing.