Lectins as membrane components of mitochondria from Ricinus communis

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Abstract
Mitochondria were isolated from developing endosperm of R. communis and were fractionated into outer membrane and inner membrane. The relative purity of the 2 membrane fractions were determined by marker enzymes. The fractions were also examined by negative-stain EM. Membrane fractions were sequentially extracted in the following way. Suspension in 0.5 M-potassium phosphate, pH 7.1; suspension in 0.1 M-EDTA (disodium salt)/0.05 M-potassium phosphate, pH 7.1; sonication in 0.05 M-potassium phosphate, pH 7.1; sonication in aqueous Triton X-100 (0.1%). The membranes were pelleted by centrifugation at 100000 g for 15 min, between each step. Agglutination activity in the extracts was investigated by using trypsin-treated rabbit erythrocytes. The addition of lactose to inner mitochondrial membrane resulted in the solubilization of part of the lectin activity, indicating that the protein was attached to the membrane via its carbohydrate-binding site. Pretreatment of the membranes with lactose before the usual extraction procedure showed that lactose could extract lectins that normally required more harsh treatment of the membrane for solubilization. Lectins extracted from inner membranes were purified by affinity chromatography on agarose gel. Polyacrylamide-gel electrophoresis of purified samples in sodium dodecyl sulfate indicated that at least part of the lectin present in inner mitochondrial membrane was identical with the R. communis agglutinin of MW 120,000.

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