PMC

Alignment of AcCa1 and AcCa2 sequences with homologs from Arabidopsis and prokaryotes. Acanthamoeba AcCa1 and AcCa2 are aligned with Arabidopsis CA1 and CAL1 along with an α-proteobacterial (Ehrlichia canis) γCA homolog and archaeal Cam (Methanosarcina thermophila). Critical amino acids described in text are referred to according to the Cam nomenclature. H42 and C137 of CA1 are denoted by * and %, respectively. Shading of columns reflects amino acid similarity of ≥ 80%.

Distinct subtypes of eukaryotic γCAs. A truncated alignment of γCA proteins demonstrates that the N-terminal regions of certain isoforms (termed Class I) are highly conserved throughout Eucarya (see text). Species abbreviations: CA1-3, Arabidopsis; Cpa, Cyanophora paradoxa; AcCa, Acanthamoeba castellanii; Tth, Tetrahymena thermophila; Ehu, Emiliania huxleyi; Pin, Phytophthora infestans; Gth, Guillardia theta. Shading of columns reflects amino acid similarity of ≥ 40%.

BN-PAGE, CI enzyme activity (panel A) and two-dimensional BN/SDS-PAGE (panel B) profiles. Panel A shows the characteristic Coomassie-stained BN-PAGE profile of Acanthamoeba mitochondrial complexes (left) and in-gel assay of these complexes (Acanthamoeba BN-PAGE lane from the same gel), identifying an NADH dehydrogenase activity at ~940 kDa (right). This band is considered to represent intact CI from Acanthamoeba. Panel B shows the protein profile observed after two-dimensional BN/SDS-PAGE of Acanthamoeba mitochondrial complexes resolved in panel A. A list of proteins identified in CI and CI* isolated from BN-PAGE bands is presented in Additional File . CI, intact complex I (940 kDa); CI* = CI subcomplex (820 kDa); CIII, complex III; CV, complex V.