Preparation and Properties of Mitochondria from Ehrlich Ascites Tumor Cells

Although the oxidative metabolism of the Ehrlich ascites turnout cell has been a matter of important theoretical discussions in recent years (cf. references I and 2), relatively few papers have appeared concerning the oxidative phosphorylation of the isolated mitoehondria of these cells (3-6). The reason for this is probably largely a technical one: the ascites cell has a very tough cell membrane and the procedure generally used for homogenizing animal tissues-with a loosely fitting Potter-Elvehjem homogenizer in isotonic s u c r o s e-i s not sufficient to break the tumour cells. In the last year we have developed a simple method to obtain aseites-eell mitochondria which seem to be fairly "intact," as judged by current biochemical criteria for mitochondria of normal tissues. We give here a short description of the isolation procedure and some of the experimental results bearing on the quality and the properties of the isolated mitoehondria. A somewhat similar procedure has recently been described by Emmelot el al. (6). The cells of an Ehrlich ascites tumour t are harvested 5 or 6 days after the inoculation of 0.2 ml. of ascites fluid in Swiss mice. The ascites fluid of 4 to l0 mice is collected in 50 ml. ice cold 0.9 per cent NaCI + 0.001 M ethylenediaminetetraacetate, pH 7.0. With all subsequent manipulations the temperature is kept below 4°C. The cells are filtered once through a loosely woven cheese cloth to remove clots, etc., and they are washed free from contaminating erythrocytes by repeated low-speed cerltrifugation in 0.25 M sucrose + 0.001 M ethylenediaminetetraacetate. The cells are then packed by centrifugation for 5 minutes at 1500 g. The packed cells are diluted to 25 ml. with redistilled water and rapidly transferred to a 30 ml. Potter-Elvehjem homogenizer with a tight-fitting pestle. The cells are mixed by 3 passes with the motor-driven pestle at low speed; after an interval of 30 seconds 3 to 5 more passes are made at high speed. The tonicity is then rapidly raised to 0.25 M by adding 2 M sucrose and the contents of the tube are thoroughly mixed. I Kindly supplied by the late Prof. B. Mendel. The homogenate obtained is diluted twofold with 0.25 M sucrose and centrifuged 5 minutes at 700 g (average). The supernatant (S0 is carefully poured off and centrifuged 10 minutes at 5600 g. The supernatant ($2) is completely poured off and the …