Neurospora crassa alpha-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties.
A method is proposed for the purification of the Neurospora crassa alpha-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. Resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on SDS-polyacrylamide gel electrophoresis as E3, E1 and E2 with respective Mr values of 54,000, 53,000 and 49,000. Catalytic properties of the purified complex were established showing the importance of divalent cations in regulating the activity level. The role of Ca2+ in particular was investigated. It was shown that Ca2+ diminishes the Km value of the N. crassa alpha-ketoglutarate dehydrogenase complex for alpha-ketoglutarate in the physiological concentration range, as previously observed for the mammalian complexes.
Bessam
H
H
Laboratoire de Biochimie, Unité Associée au CNRS No. 203, Faculté des Sciences, Université de Rouen Haute-Normandie, Mont-Saint-Aignan, France.
Mareck
A M
AM
Foucher
B
B
D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D002413:Cations, Divalent; D003065:Coenzyme A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007655:Ketoglutarate Dehydrogenase Complex / Q000302:isolation & purification / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D013696:Temperature; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D002413:Cations, Divalent; D007656:Ketoglutaric Acids; D009243:NAD; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D008274:Magnesium; D013835:Thiamine Pyrophosphate; D003065:Coenzyme A; D002118:Calcium