Topography and organisation of proteins and glycoproteins in rat liver mitochondrial membranes

The basic objective of this thesis is to develop our understanding of the topography, organisation and function of mitochondrial membrane proteins/glycoproteins to a level comparable to the red blood cell plasma membrane. Initial characterisation of the isolated standard mitochondrial fraction (M1) or sucrose density purified material (PM1), involves assessment of the extent of contamination with other subcellular fractions. M1 preparations contain 5.0% acid phosphatase (lysosomes), 5.4% glue ose-6-phosphate (microsomes) and 4.4% 5'-nucleotidase (plasma membrane), while analogous values for the PM1 fractions are 1,1%, 1.7% and 0.6% respectively. Integrity of the outer membrane in isolated mitochondria is monitored by an assay using exogeneous cytochrome c as substrate for cytochrome c oxidase, an inner membrane protein complex. Estimates of the degree of damage to the outer membrane reveal 3-5% breakage in the M1 mitochondria and 11-17% in PM mitochondria, 1 Optimum procedures for subfractionation of mitochondria into inner and outer membranes, and a soluble fraction are developed employing sonication and sucrose density gradient analysis. Minimal cross-contamination (less than 10%) of individual fractions is achieved as assessed by specific marker enzymes, namely cytochrome c oxidase for the inner membrane, monoamine oxidase for the outer membrane and adenylate kinase for the soluble fraction. Analysis of rat liver mitoctiondria pre-labelled in vivo with D-[6-3H]-glucosamine confirm that 70-80% of the carbohydrate is released in a soluble form on disruption of the organelle, the remainder being distributed equally between inner and outer membranes. The bulk of the carbohydrate is associated with protein rather than lipid. Treatment of rat liver mitochondria with low levels of digitonin (0.01mg/mg protein) releases approx. 50% of the glucosamine-labelled mitochondrial glycoprotein, without destroying the integrity of the outer membrane. Immuno- precipitation of these extrinsic components with Con A and anti-Con A serum reveals 4 components, including a major doublet of 40,000 and 48,000 mol. wt. Direct agglutination and binding studies with[125I]-WGA demonstrate the presence of integral carbohydrate on the external surfaces of mitochondrial inner and outer membranes. (Abstract shortened by ProQuest.)