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Selective labeling of beef heart cytochrome oxidase subunit III with eosin-5-maleimide
Abstract:
Cytochrome c oxidase has been isolated from beef heart mitochondria and labeled with the fluorochrome eosin-5-maleimide (EMA) after pretreatment with mersalyl. On SDS-polyacrylamide gels, EMA fluorescence and absorption occurred at a single band corresponding to subunit III. Since only Cys 115 of the two cysteinyl residues of subunit III had been shown to be reactive towards water-soluble SH-reagents, it was concluded that this residue was the one labeled by EMA. The EMA/enzyme ratio was about 1. Gel filtration experiments have shown that upon treatment with dicyclohexylcarbodiimide, subunit III was loosened from the complex; this result suggests that the inhibitory effect of dicyclohexylcarbodiimide on the H+-translocation activity may be related to such a phenomenon.
Insights
Beef heart cytochrome c oxidase subunit III was labeled with eosin-5-maleimide (EMA) at Cys 115. Dicyclohexylcarbodiimide treatment loosened subunit III, suggesting a role in H+-translocation inhibition.
Area of Science:
- Biochemistry
- Mitochondrial Function
- Enzyme Kinetics
Background:
- Cytochrome c oxidase is a key enzyme in the mitochondrial electron transport chain.
- Understanding subunit interactions is crucial for elucidating enzyme function and regulation.
Purpose of the Study:
- To identify the specific site of eosin-5-maleimide (EMA) labeling on beef heart cytochrome c oxidase.
- To investigate the role of subunit III in the enzyme complex and its interaction with dicyclohexylcarbodiimide (DCCD).
Main Methods:
- Isolation and purification of beef heart cytochrome c oxidase.
- Labeling with eosin-5-maleimide (EMA) after mersalyl pretreatment.
- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for fluorescence and absorption analysis.
- Gel filtration chromatography to assess subunit dissociation.
Main Results:
- EMA fluorescence and absorption localized to a single band corresponding to subunit III on SDS-PAGE.
- The reactive residue was identified as Cys 115, based on prior knowledge of cysteinyl residue reactivity.
- The EMA/enzyme ratio was determined to be approximately 1.
- Gel filtration showed that dicyclohexylcarbodiimide (DCCD) treatment loosened subunit III from the complex.
Conclusions:
- Cys 115 of subunit III is the primary site for EMA labeling in beef heart cytochrome c oxidase.
- DCCD-induced dissociation of subunit III suggests its involvement in maintaining the structural integrity of the enzyme complex.
- The observed loosening of subunit III may explain the inhibitory effect of DCCD on proton translocation activity.