Subunit specific monoclonal antibodies show different steady-state levels of various cytochrome-c oxidase subunits in chronic progressive external ophthalmoplegia.
Monoclonal antibodies recognizing the mitochondrially encoded subunits I and II, and the nuclear-encoded subunits IV, Va, Vb and VIc of human cytochrome-c oxidase were generated. These antibodies are highly specific and allow the assessment of subunit steady-state levels in crude cell extracts and tissue sections. In the experimental human cell line 143B206, which is devoid of mitochondrial DNA, immunovisualization with the antibodies revealed that the nuclear-encoded subunits IV and Va were present in amounts close to that of the parental cell line despite the absence of the mitochondrially encoded subunits. In contrast, the nuclear-encoded subunits Vb and VIc were severely reduced in cell line 143B206, suggesting that unassembled nuclear-encoded subunits are degraded at different rates. In skeletal muscle sections of a patient with chronic progressive external ophthalmoplegia known to harbor the 'common deletion' in a subpopulation of her mitochondrial DNA, most cytochrome-c oxidase activity negative fibers had greatly reduced levels of subunits I, II, Va, Vb and VIc of cytochrome-c oxidase. The steady-state level of subunit IV, however, was less affected. This was particularly evident in cytochrome-c oxidase activity negative fibers with accumulated mitochondria ('ragged-red' fibers) where immunodetection with anti-subunit IV resulted in intense staining. The data presented in this paper demonstrate that the battery of monoclonal antibodies can be employed for diagnostic purposes to analyze steady-state levels of mitochondrially and nuclear-encoded subunits of cytochrome-c oxidase.
Taanman
J W
JW
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA. taanman@rfhsm.ac.uk
Burton
M D
MD
Marusich
M F
MF
Kennaway
N G
NG
Capaldi
R A
RA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics; D003576:Electron Transport Complex IV / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D005260:Female; D006801:Humans; D051379:Mice; D008929:Mitochondria, Heart / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology*; D018482:Muscle, Skeletal / Q000201:enzymology; D017246:Ophthalmoplegia, Chronic Progressive External / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D004272:DNA, Mitochondrial; D003576:Electron Transport Complex IV