The cytochromes of Acanthamoeba castellanii

Abstract

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).

Oxygen induction of a novel fatty acid n−6 desaturase in the soil protozoon, Acanthamoeba castellanii

“…Assay of NADH : cytochrome b & oxidoreductase activity was based on the methods for microsomal electron transport chain activities in yeast, T. pyriformis and A. castellanii [29][30][31]. Cytochrome b & levels were measured using a dual-wavelength spectrophotometer (Hitachi-Perkin Elmer ; model 557).…”

Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii

“…Spectral evidence suggests the presence of b-, cand a-type cytochromes in mitochondria of A. castellanii (Lloyd & Griffiths, 1968;Edwards et al, 1977). In difference spectra, there is evidence for two cyanide-reacting cytochromes (Edwards et al, 1977), one of whichmay be cytochrome a3.…”

“…Difference spectra (Chance, 1957) were recorded at 77 K in a Unicam SP. 1800 spectrophotometer (Edwards & Lloyd, 1973;Edwards et al, 1977); reduction of the sample cuvette was by sodium dithionite and oxidation of the reference cuvette by potassium ferricyanide. Steady-state reduction of cytochromes (Chance & Williams, 1956), in the presence or absence of inhibitors, was measured on mitochondrial preparations suspended in respiratory medium.…”

“…In difference spectra, there is evidence for two cyanide-reacting cytochromes (Edwards et al, 1977), one of whichmay be cytochrome a3. Also, there are a 203 S. W. EDWARDS AND D. LLOYD number of CO-reacting pigments which participate in whole-cell respiration, and there is evidence for an oxidase with a low affinity for oxygen (Edwards et al, 1977).…”

“…A preliminary account of this work has been presented (Edwards & Lloyd, 1977c). Experimental Maintenance, growth and harvesting of the organisms Acanthamoeba castellanii was maintained and grown axenically, with shaking at 30°C, exactly as described previously (Edwards et al, 1977 Disruption of the organism and isolation of mitochondria Cells were washed once with lOvol. of 50mM-MgCl2, pH7.4, and resedimented by centrifugation at 3000rev./min for 1.5min in the 6 x 250ml head of an MSE 18 centrifuge.…”