Unusual properties of mitochondria from the human term placenta are caused by alkaline phosphatase

Human placental mitochondria prepared by a new isolation procedure exhibit low but well coupled rates of state 3 respiration with different substrates (succinate: 32.3 nmol O2/mg/min, RCI = 4.4; pyruvate: 12.6 nmol O2/mg/min, RCI R = 4.2; palmitoylcarnitine: 16.6 nmol O2/mg/min, RCI R = 4.9). The addition of the uncoupler FCCP increased the respiratory rates (succinate: 40.7 nmol O2/mg/min; pyruvate: 21.2 nmol O2/mg/min: palmitoylcarnitine: 25.4 nmol O2/mg/min). The low respiratory rates correlate well with a low capacity of the respiratory chain as shown by the specific contents of cytochrome c (0.15 nmol/mg), cytochrome b (0.19 nmol/mg) and cytochrome oxidase (0.14 nmol/mg) as well as with the low content of adenine nucleotides (2.71 nmol/mg). These data together with the finding of high activities of alkaline phosphatase (2.2 U/mg) support the view that human placental mitochondria are contaminated with nonmitochondrial membranes. Since it was not possible to obtain functionally intact mitochondria with negligible activities of alkaline phosphatase the influence of this enzyme on the extramitochondrial adenine nucleotide turnover was investigated. Alkaline phosphatase splits phosphate from ATP, ADP and AMP with different rates resulting in an intermediate accumulation of AMP. Mitochondrial adenylate kinase (0.16 U/mg) regenerated ADP from AMP and ATP resulting in drastically decreased ADP/O ratios and prolonged state 3 respirations. Inhibiting the adenylate kinase with diadenosine pentaphosphate the ADP regeneration from AMP and ATP was suppressed which, in turn, enhanced the ADP/O ratios. In the absence of magnesium ions, if both the alkaline phosphatase and the adenylate kinase are inhibited normal ADP/O ratios and state 3-state 4 transitions can be observed. Under these conditions, human placental mitochondria showed normal properties comparable to those of mitochondria from other tissues with the only exception of low specific activities.