Affinity chromatography isolation of human cytochrome oxidase and small-scale Western immunoblot probing of the enzyme complex in mitochondrial cytopathy patients.
Isolation of human cytochrome oxidase by a one-step affinity chromatography procedure on a Sepharose 4B-ferrocytochrome c matrix following solubilization with the nonionic detergent laurylmaltoside yields an enzyme isolate of adequate purity for producing polyclonal antisera. Such an antiserum produced a distinctive immunoreactive profile in Western immunoblot studies to that reported using the enzyme isolated with ionic detergents. A sensitive and highly reproducible Western immunoblotting method is described for probing mitochondrial fractions prepared from small frozen skeletal muscle biopsies with an antiserum against the human placenta cytochrome oxidase. Application of this method to mitochondrial cytopathy patients with partial cytochrome oxidase deficiency shows that the detected subunits are synthesized in these patients.
Trounce
I
I
Department of Neurology, St. Vincent's Hospital, Melbourne, Australia.
Byrne
E
E
Dennett
X
X
Chen
W W
WW
Marzuki
S
S
D015153:Blotting, Western / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D030401:Cytochrome-c Oxidase Deficiency; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008931:Mitochondria, Muscle / Q000201:enzymology; D009135:Muscular Diseases / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011487:Protein Conformation
D003576:Electron Transport Complex IV