Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method.
A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.
D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007655:Ketoglutarate Dehydrogenase Complex / Q000302:isolation & purification*; D007658:Ketone Oxidoreductases / Q000302:isolation & purification*; D008722:Methods; D009206:Myocardium / Q000201:enzymology*; D011092:Polyethylene Glycols; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification*; D014461:Ultracentrifugation
D011768:Pyruvate Dehydrogenase Complex; D011092:Polyethylene Glycols; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex