Cytochrome Oxidase of Cactus

The present autlhors have isolated a particleassociated cytochrome oxidase from the phylloclades of cactus plant and studied its kinetics. To our knowledge, this is the first report on cytoclhrome oxidase from a crassulacean plant. Tender phylloclades of Nopalea dejecta SalmDyck were ground with 5 volumes of chilled medium in a Waring Blendor operated first at low speed for 25 seconds and then at maximum speed for 5 seconds. The medium, essentially that of Wiskich and Bonner (10), consisted of: 0.32 M mannitol, 0.25 M sucrose, and 5 mM EDTA in 0.1 M tris buffer adjusted to pH 7.8. The suspension was squeezed through 2 layers of muslin into a chlilled container and the filtrate (pH 7.0-7.2) subjected to differential centrifugation. The mitochondria sedimenting between 1600g and 15,00(g during 30 minutes were collectecd and washed twice by suspending in a solution of 0.32 M mannitol and of 0.25 M sucrose and recentrifuging for 30 minutes at 15,000g. W'Iith the aid of a loosely fitting Potter-Elvehjem homogenizer, the final preparation was dispersed in sufficient 0.5 M mannitol to give a suspension equivalent to 2 g fresh tissue per ml., The protein content as determined by the method of Lowry et al. (4) was 1.4 to 2.0 mg per ml suspens.ion. This method of isolation of mitochondria yielded the most active preparation from the point of view of oxidative phosphorylation. The preparation was free from ascorbate oxidase. The spectrophotometric assay was carried out according to Cooperstein and Lazarow (1), at the optimunm pH of 7.5. The initial rate of the reatction was calculated according to Yonetani (11). Tihe manometric assay of enzyme activity was according to Lieberman (3) with ascorbic acid as the hydrogen donor, but with the difference that ATP was not added and that phosphate concentration was higher 1(0.067 M ). Tihe rate of the reactionl was liniear from the tenth minute to the end of the hour. The activity was calculate(d from the total oxygen uptake at the end of 60 minutes. The oxvgen uptake due to cytochrome oxidase action was obtained by subtracting the value for the autoxidation of ascorbate. The kinetics of cactus cytochrome oxidase in the spectrophotometric assays was of the first order with respect to substrate at all concentrations tested