Isolation of complex III from various mitochondria. Comparison of the structural and functional properties of the preparations from beef heart, calf liver and Neurospora crassa.

Abstract

Active complex III was isolated by an improved procedure from beef heart mitochondria, from Neurospora crassa mitochondria and for the first time from mitochondria originating from mammalian tissue other than heart, i.e. calf liver. The described procedure consists of differential extraction of the respective mitochondria, hydroxyapatite chromatography and, finally, either gel- or affinity chromatography. The preparations contain the well known prosthetic groups, i.e. 6-8 mumol b-type heme, 3-4 mumol c-type heme and 5-8 mumol non-heme iron per g of protein. The preparations from beef heart and from calf liver mitochondria are indistinguishable in their subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis, whereas the preparation from Neurospora crassa mitochondria is clearly different. The phospholipid content of all preparations is rather low, amounting to about 100 mumol/g protein. The molar catalytic activity of ubiquinol-9-cytochrome c reductase at 25 degrees C amounts to 50s-1 for the N. crassa complex III and 70-100s-1 for the bovine complexes. After reincorporation into phospholipid vesicles, all preparations how tight coupling between electron transfer from ubiquinol to cytochrome c and proton translocation across the phospholipid bilayer.