Broad-zone active-enzyme chromatography. Keto-acid dehydrogenases as associating systems.
We have extended the method of active-enzyme chromatography to include the use of broad zones of enzyme. This allows examination of interacting systems in a way formally analogous to sedimentation velocity so that simulation of the observed activity profiles is possible. The method has been applied using pyridine nucleotide-linked active enzyme assays. At the concentrations presently accessible by this technique, hexokinase and glucose-6-phosphate dehydrogenase, both associating systems, show single symmetrical boundaries, as does isolated diaphorase, while pyruvate and alpha-ketoglutarate dehydrogenases show more complex patterns, with the position of the reaction boundary for diaphorase activity being dependent on enzyme concentration.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002845:Chromatography / Q000379:methods; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D005954:Glucosephosphate Dehydrogenase; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase; D006593:Hexokinase