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.