PROTEIN SYNTHESIS IN MITOCHONDRIA. 4. PREPARATION AND PROPERTIES OF MITOCHONDRIA FROM KREBS II MOUSE ASCITES-TUMOUR CELLS

Abstract

Methods of disrupting Krebs II mouse ascites-tumor cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0.3 [image]-sucrose. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succinoxidase was measured in the various cell fractions after separation by differential centrifuging. The mitochondrial fraction sedimented at 9500 g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500 g pellet contains other material besides mitochondria. Krebs-cell mitochondria contain up to 5 times as much RNA as do liver mitochondria. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.