Adsorptive immobilization of submitochondrial particles on concanavalin A Sepharose-4B

Submitochondrial particles (SMPs) prepared from beef liver mitochondria were immobilized on concanavalin A Sepharose-4B (Con A-Sepharose). The process of immobilization was optimized by choosing an appropriate buffer system containing Mn2+ and Ca2+. Adsorption of SMPs on Con A-Sepharose was found to be reversible process, nonelectrostatic in nature, and responsive to the presence of methyl alpha-d-glucopyranose and alpha-d-mannose. The involvement of membrane glycoproteins in the adsorption process was thus demonstrated. Further analysis of the data obtained on competition of binding by sugar molecules provided competition constants reflecting the potency of inhibition by each individual carbohydrate. Positive-cooperative interactions for binding to the matrix were observed especially at high concentrations of SMPs. The immobilized preparations were used successfully in continuous catalytic transformations involving succinate-cytochrome c reductase (SCR) and enzyme complex of the inner-mitochondrial membrane. Best results were obtained when such operations were carried out at 37 degrees C.