Summary
Cytoplasmic and mitochondrial isozymes of NADP+-dependent isocitrate dehydrogenase were purified from kidney and heart tissue of an inbred strain of mice. The cytoplasmic isozyme was purified from kidney of DBA/2J mice by means of a four-step procedure which included affinity chromatography with an 8-(6-aminohexyl)-amino-NADP+-Sepharose column. The heart mitochondrial isozyme of DBA/2J mice was purified by a two-step procedure involving the use of 8-(6-aminohexyl)-amino-AMP-Sepharose and 8-(6-aminohexyl)-amino-NADP+-Sepharose columns. The specific activity of the homogeneous cytoplasmic and mitochondrial isozymes was 40 units/mg and 45 units/mg, respectively. Native and subunit molecular weights of these two isozymes were determined by chromatography on Sephadex G-100, G-150 and G-200 Superfine and polyacrylamide gel electrophoresis. Both isozymes were found to be dimers with the subunit molecular weight of approximatively 35,000. The sedimentation coefficients were determined to be 5.9 and 6.1 for the mitochondrial and cytoplasmic isozyme, respectively. The amino acid compositions of these two isozymes revealed distinct differences in arginine and proline contents. A modified procedure regarding the use of affinity columns for the purification of the weakly bound enzymes is also discussed.
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Pegoraro, B., Yuan, J.H. & Lee, CY. Purification and structural properties of isozymes of isocitrate dehydrogenase from the mouse. Mol Cell Biochem 23, 177–184 (1979). https://doi.org/10.1007/BF00219456
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DOI: https://doi.org/10.1007/BF00219456