Ultrastructural and biochemical studies of mitoplasts and outer membranes derived from French-pressed mitochondria: Advances in mitochondrial subfractionation

Abstract

Rapid separation of mitoplasts and outer membranes of rat liver mitochondria has been accomplished by extrusion of mitochondrial suspensions through a French pressure cell. Preparations were monitored using established micro-sampling techniques to determine the extent of outer membrane release, the degree of homogeneity of the mitochondrial subfractions, and the affect of French pressing on mitochondrial ultrastructure. The distribution of “marker enzymes,” monoamine oxidase (MAO), cytochrome oxidase (Cyt Ox), malate dehydrogenase (MDH), and adenylate kinase (ADK) was similar to that obtained when mitochondrial subfractionation was carried out using digitonin. For the first time a strictly physical method of removing the outer mitochondrial membrane which has yielded a mitoplast fraction retaining both acceptor control and a condensed configuration characteristic of the inner compartment of freshly isolated mitochondria is reported. Similar separation of the same components of rat heart mitochondria has also been accomplished. Thus, by this method well-defined mitochondrial subfractions, uncontaminated with detergents, have been isolated from two mammalian tissues. Digitonin treatment of mitoplasts prepared by the technique reported here resulted in the conversion of the condensed configuration to that of the digitonin-derived mitoplast. In addition, further insight into the ultrastructural nature of inner and outer membrane contact phenomena has been established.