Studies on the binding of concanavalin A to rat liver mitochondria.

The binding of concanavalin A (con A) to intact and detergent-solubilized fractions of rat liver mitochondria has been studied quantitatively using a turbidimetric agglutination assay and by a radioactive binding procedure employing [acefyl-3H]con A. The reaction appears to involve oligosaccharide residues associated with glycoproteins of the mitochondrion since glycosides such as methyl-c-u-D-mannopyranoside and methyl-ar-o-glucopyranoside, which are specifically bound by con A, will inhibit this interaction. Solvent extraction (10% water in acetone) did not affect the ability of mitochondria to bind [acetyZ-3H]con A. Based on gel filtration studies of Triton X-100 (1%) solubilized preparations of mitochondria, the majority (70%) of the con Areactive components have molecular weights of 40,000 to 80,000. Since approximately 40 % of the total [acefyl3H]con A-binding capacity can be demonstrated using intact mitochondria, it appears that a considerable number of binding sites are located on the outer surface of the mitochondrial outer membrane. Seventy per cent of the con A-reactive components are released in soluble form together with most (85%) of the intracristate marker enzyme adenylate kinase by treatment with 0.108 mg of digitonin per mg of mitochondrial protein. The remainder of the con A-binding material is distributed equally between the inner and outer mitochondrial membranes. When the digitonin-solubilized intracristate space fraction is subjected to polyacrylamide gel electrophoresis five periodic acid-Schiff’s base staining bands are observed. When the soluble, intracristate protein fraction is subjected to trypsin digestion or periodic acid oxidation, extensive destruction of [acetyZ-3H]con A-binding activity is observed. These observations indicate that con A may be a useful probe for investigating the function and distribution of glycoproteins in the mitochondrion.