Plasmodium berghei: partial purification and characterization of the mitochondrial cytochrome c oxidase.
Mitochondria from a rodent malarial parasite (Plasmodium berghei) were successfully purified by differential centrifugation and 22% Percoll density gradient separation. The purified mitochondria from the erythrocytic stages of the parasite had a density of 1.05 and were found to be heterogeneous by transmission electron microscopy and rhodamine 123 fluorescence microscopy. Three marker enzymes, dihydroorotate dehydrogenase, cytochrome c reductase, and cytochrome c oxidase, were assessed during the organelle separation. Purification of cytochrome c oxidase was carried out from the purified mitochondria by using combination techniques of detergent solubilization and reduced cytochrome c-agarose affinity chromatography. The 560-fold purified enzyme with 3.6% yield was obtained and it had low catalytic efficiency with a kcat/Km of 5.9 x 10(-5) M-1 x min-1. The native form of the enzyme, determined by a gel filtration column on fast protein liquid chromatography, was found to be an oligomeric structure with a minimal molecular weight of 670 kDa. The malarial enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then compared to the enzyme obtained from host liver cells. These results suggested that the partially purified enzyme from the parasite was not different from its host mammalian cells. The importance of the enzyme in the erythrocytic phase of the parasite is discussed as a part of a simple electron transport system in mitochondrion linked to limited oxygen utilization and pyrimidine de novo biosynthesis.
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
J
J
Krungkrai
S R
SR
Krungkrai
A
A
Bhumiratana
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D010962:Plasmodium berghei / Q000201:enzymology*
D003576:Electron Transport Complex IV