Mitochondria from human term placenta. I. Isolation and assay conditions for oxidative phosphorylation

Human term placental mitochondria were resolved by differential centrifugation into three fractions, heavy mitochondria, light mitochondria and a third, less dense fraction. Approximately equal amounts of mitochondrial protein were found in the three fractions. These mitochondrial preparations differed in physical properties. ATPase and "ADPase" content and oxidative capacities. Assay conditions were developed which permitted the polarographic measurement of respiration and coupled phosphorylation carried out by all three mitocondrial preparations despite the variable nucleotide-phosphate phosphatase activities present. With heavy mitochondria, rates of respiration were consistently higher than those previously reported for unfractionated placental mitochondria. Respiratory control ratios were comparable to those of mitochondria from other steroid hormone-producing endocrine tissues and ADP/O ratios approaching the theoretical maxima were obtained. Both lighter placental mitochondrial fractions displayed somewhat lower respiration rates and respiratory control but their primary defect was a selective uncoupling of the third site of energy conservation. Modification of isolation procedures were evaluated in terms of quantitative yield and functional activity of the three fractions.