Immunological identification of the alternative oxidase of Acanthamoeba castellanii mitochondria

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 cyanideresistant respiration.

ATP-sensitive Potassium Channel in Mitochondria of the Eukaryotic Microorganism Acanthamoeba castellanii

“…Cell Culture and Isolation of Mitochondria-The soil amoeba A. castellanii, strain Neff, was cultured as described by Jarmuszkiewicz et al (22). Trophozoites of the amoeba were collected between 44 and 48 h following inoculation at the middle exponential phase (at a density of about 4 -6 ϫ 10 6 cells/ ml).…”

“…castellanii and higher plants share several common features at the level of the respiratory chain of the inner mitochondrial membrane, such as the presence of an alternative cyanide-resistant ubiquinol oxidase and nonphosphorylating rotenoneinsensitive internal (matrix face) and external (cytosolic face) NADH dehydrogenases (22)(23)(24). On the contrary, recent studies on the translocase of the outer membrane (TOM) complex of A. castellanii mitochondria suggest that it could be rather similar to the animal-fungal TOM proteins (25).…”

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

“…Trophozoites of the amoeba were collected between 46 and 48 h following inoculation at the late exponential phase at a density of approximately 4-5×10 6 cells/ml. The mitochondria were isolated and purified on a self-generating Percoll gradient (29 %) as previously described (Jarmuszkiewicz et al 1997). The presence of 0.3 % bovine serum albumin (BSA) in isolation media allowed the endogenous FFA to be chelated from the mitochondrial suspension.…”

Impact of oxidative stress on Acanthamoeba castellanii mitochondrial bioenergetics depends on cell growth stage

“…Mitochondria were isolated by differential centrifugation and purified on a self-generating Percoll gradient (30%) as described previously (Jarmuszkiewicz et al 1997). The depletion of endogenous free fatty acids in the mitochondrial preparations was ensured by the presence of 0.4% fatty acidfree bovine serum albumin (BSA) in the isolation media except for the last mitochondrial washing (Jarmuszkiewicz et al 1999).…”

“…Under axenic non-pathogenic conditions, A. castellanii has been used as a model organism to study mitochondrial energydissipating systems, such as a cyanide-resistant alternative oxidase (AcAOX) (Jarmuszkiewicz et al 2005a;Woyda-Ploszczyca et al 2009), an ATP-sensitive potassium channel , and an uncoupling protein (AcUCP) (Jarmuszkiewicz et al 2010). The mitochondria of A. castellanii contain a plant-type respiratory chain with additional (in addition to the four classical) electron carriers in the form of external and internal NADH dehydrogenases and an alternative cyanide-resistant quinol oxidase (AOX) that consumes mitochondrial reducing power without energy conservation in the proton electrochemical gradient (Jarmuszkiewicz et al 1997). We have shown that in A. castellanii, as in plant mitochondria (Popov 2003;Vercesi et al 2006), the two mitochondrial energy-dissipating systems, AcAOX and AcUCP, may play a role in the energetic status of the cell by decreasing the yield of ATP synthesis and in attenuating ROS production (Czarna and Jarmuszkiewicz 2005;Czarna et al 2007).…”