Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography.
1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which  are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.
D000473:Alkanes; D000818:Animals; D002846:Chromatography, Affinity; D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification*; D007527:Isoenzymes / Q000302:isolation & purification*; D008024:Ligands; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D009994:Osmolar Concentration; D011485:Protein Binding; D012685:Sepharose; D013329:Structure-Activity Relationship; D013552:Swine; D013696:Temperature; D008063:Thioctic Acid
D000473:Alkanes; D007527:Isoenzymes; D008024:Ligands; D009243:NAD; D008063:Thioctic Acid; D012685:Sepharose; D008058:Dihydrolipoamide Dehydrogenase