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Immunological identification of the alternative oxidase of Acanthamoeba castellanii mitochondria
W Jarmuszkiewicz1, A M Wagner, M J Wagner
1Department of Bioenergetics, Institute of Molecular Biology and Biotechnology, Poznan University, Poland. wiesiaj@vm.amu.edu.pl
Abstract:
Mitochondria of the protozoa Acanthamoeba castellanii possess a cyanide-insensitive oxidase cross-reacting with monoclonal antibodies raised against the plant alternative oxidase. Immunoblotting revealed three monomeric forms (38, 35, and 32 kDa) and very low amounts of a single 65 kDa dimeric form. Cross-linking studies suggest that while in plants the alternative oxidase occurs as a dimer, in amoeba it functions as a monomer. Immunologically detectable protein levels change with the age of amoeba cell culture. Increased amounts of the 35 kDa protein are accompanied by an increase in the activity of cyanide-resistant respiration.
Insights
Mitochondria in Acanthamoeba castellanii contain a cyanide-insensitive oxidase similar to plant alternative oxidase. This oxidase functions as a monomer in amoeba, unlike its dimeric form in plants, and its levels correlate with respiration activity.
Area of Science:
- Biochemistry
- Cell Biology
- Protozoology
Background:
- Mitochondria are crucial for cellular respiration.
- Alternative oxidases (AOX) provide a cyanide-insensitive respiratory pathway.
- The structure and function of AOX can vary across different organisms.
Purpose of the Study:
- To investigate the presence and characteristics of cyanide-insensitive oxidase in Acanthamoeba castellanii mitochondria.
- To compare the structure and function of this oxidase with plant alternative oxidase.
- To explore the relationship between oxidase levels and respiratory activity in Acanthamoeba.
Main Methods:
- Immunoblotting using antibodies against plant alternative oxidase.
- Mitochondrial isolation and protein extraction from Acanthamoeba castellanii.
- Cross-linking studies to determine protein oligomeric state.
- Measurement of cyanide-resistant respiration activity.
Main Results:
- Acanthamoeba mitochondria possess a cyanide-insensitive oxidase that cross-reacts with antibodies to plant AOX.
- The amoeba oxidase exists primarily as 38, 35, and 32 kDa monomeric forms, with minimal 65 kDa dimeric form.
- Cross-linking suggests the amoeba oxidase functions as a monomer, contrasting with the plant dimeric form.
- Protein levels of the 35 kDa form increase with cell culture age, correlating with enhanced cyanide-resistant respiration.
Conclusions:
- Acanthamoeba castellanii harbors a monomeric alternative oxidase in its mitochondria.
- This amoeba AOX differs structurally from its plant counterpart.
- The abundance of the 35 kDa AOX monomer is linked to increased cyanide-resistant respiratory capacity in Acanthamoeba.