Rat liver mitochondria were fragmented by a combined technique of swelling, shrinking, and sonication. Fragments of inner membrane were separated by density gradient centrifugation. They differed in several respects: electronmicroscopic appearance, phospholipid and cytochrome contents, electrophoretic behaviour of proteins and enzymatic activities.
Three types of inner membrane fractions were isolated. The first type is characterized by a high activity of metal chelatase, low activities of succinate-cytochrome c reductase and of glycerolphosphate dehydrogenase, as well as by a high phospholipid content and low contents of cytochromes aa3 and b.
The second type displays maximal activities of glycerolphosphate dehydrogenase and metal chelatase, but contains relatively little cytochromes and has low succinate-cytochrome c reductase activity.
The third type exhibits highest succinate-cytochrome c reductase activity, a high metal chelatase activity and highest cytochrome contents. However, this fraction was low in both glycerolphosphate dehydrogenase activity and phospholipid content. This fraction was also richest in the following enzyme activities: cytochrome oxidase, oligomycin-sensitive ATPase, proline oxidase, 3-hydroxybutyrate dehydrogenase and rotenone-sensitive NADH-cytochrome c reductase.
Amino acid incorporation in vitro and in vivo in the presence of cycloheximide occurs predominantly into inner membrane fractions from the second type.
These data suggest that the inner membrane is composed of differently organized parts, and that polypeptides synthesized by mitochondrial ribosomes are integrated into specific parts of the inner membrane.