Abstract
An improved method is described for the isolation of Ehrlich-tumour mitochondria. The ascites cells are pre-stressed by swelling in hypotonic solution and are then disrupted into pure water by osmotic shock. Addition of concentrated sucrose solution to the homogenate immediately after lysis gives a 0.25 M-sucrose suspension, from which mitochondria can be isolated by differential centrifuging in the customary manner. The method affords clean mitochondria which are free of adhering cytoplasmic material and are functionally intact, as evidenced by their oxidative phosphorylation activity shown with succinate, [alpha]-oxoglutarate and L-glutamate as substrates. Studies with succinate and ascorbate as substrates suggest that the cytochrome c phosphorylation site is uncoupled to a considerable extent in Ehrlich ascites-cell mitochondria. Ehrlich ascites-cell mitochondria exhibit a pronounced diphosphopyridine nucleotide requirement for maximal oxidative phosphorylation activity with [alpha]-oxoglutarate and L-glutamate as substrates (Wenner-Weinhouse effect). Evidence suggests that the diphosphopyridine nucelotidase inhibitor, nicotinamide, may possibly be more effective than diphosphopyridine nucleotide itself in raising the P:O ratio.