Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria.
A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.
D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007655:Ketoglutarate Dehydrogenase Complex / Q000302:isolation & purification* / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000302:isolation & purification*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D011092:Polyethylene Glycols; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification* / Q000378:metabolism
D011768:Pyruvate Dehydrogenase Complex; D011092:Polyethylene Glycols; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex