Summary
Biochemical properties of cytoplasmic and mitochondrial isozymes of isocitrate dehydrogenase from DBA/2J mice were compared under various experimental conditions. These included Km determinations, coenzyme specificity, pH dependence, urea, iodoacetate and thermal inactivation and fluorescence titration studies. From these comparative studies each isozyme was found to have distinct coenzyme specificity, thermal stability and sensitivity to alkylation. In the case of the cytoplasmic isozyme, both NADP+ and isocitrate protect the enzyme against thermal denaturation but not iodoacetate inactivation. On the contrary, neither NADP+ nor isocitrate protects the mitochondrial enzyme against thermal or iodoacetate inactivation. Both isozymes exhibit similar fluorescence properties. NADP+ and NADPH, but not isocitrate, cause quenching of protein fluorescence. Enhancement of coenzyme fluorescence and protein energy transfer was observed when either isozyme was added to NADPH solutions. Further addition of isocitrate or isocitrate-Mg++ to a NADPH-enzyme solution caused a decrease of the enhancement of coenzyme fluorescence and protein energy transfer, but not quenching of protein fluorescence, indicating the formation of a ternary complex. This observation precludes the mechanism of mutual exclusion between NADPH and isocitrate in the active site of the enzyme.
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Abbreviations
- IDH:
-
isocitrate dehydrogenase
- NHDP+ :
-
nicotinamide-hypoxanthine dinucleotide phosphate
- TNADP+ :
-
thionicotinamide-adenine dinucoleotide phosphate
- AcPyADP+ :
-
3-acetylpyridine-adenine dinucleotide phosphate
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Pegoraro, B., Lee, CY. Comparative biochemical studies of isozymes of isocitrate dehydrogenase from the mouse. Mol Cell Biochem 24, 3–8 (1979). https://doi.org/10.1007/BF00220188
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DOI: https://doi.org/10.1007/BF00220188