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PubMed1979

Functional investigations of isolated mitochondria under steady-state conditions by means of a perfusion technique.

It was observed that borosilicate glass prefilters have a high absorptive capacity for isolated rat-liver mitochondria and that this binding does not need any kind of chemical procedure. This observation has led to the development of a perifusion technique for isolated rat-liver mitochondria. During perifusion the mitochondria are immobilized on a prefilter. Their morphological and functional intactness is conserved. During the course of the perifusion no loss of marker enzymes (adenylate kinase and malate dehydrogenase) can be detected. The respiration rates in the controlled and the active state are similar to those observed in a conventional closed polarographic vessel. The respiratory control is maintained for more than 30 min. With the perifusion technique it is possible to adjust respiration rates to stationary steady states between the controlled and the active state. It was shown that the control of respiration by the extramitochondrial ATP/ADP ratio is independent of the succinate concentration in the range of 1 to 10 mM.

Adenosine Diphosphate↗

PubMed1976

Control of oxidative phosphorylation by the extra-mitochondrial ATP/ADP ratio.

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate+rotenone and glutamate+malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized. The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.

Adenosine Diphosphate↗