Respiration-independent Binding of SR²⁺ to Bean Mitochondria

Abstract

ugation as previously described (7) in a medium containing: 0.4 M mannitol, 1 mg/ml bovine serum albumin 50 mm Tricine buffer (pH 7.5), and 2.0 mm Na-EDTA. The mitochondria were washed in a medium free of bovine serum albumin and Na-EDTA during the final centrifugation. Respiration-independent binding of Sr" was measured in a reaction mixture of 3.2 ml containing: 0.4 M mannitol, 50 mm Tricine buffer (pH 7.5), 5 AtM rotenone, 2.4 Mm antimycin A, and concentrations of Sr2+, as indicated in the legends. Under these conditions no respiration could be detected. After incubation for 90 sec, the mitochondria were harvested by centrifugation in the cold at 27,000g for 5 min, and aliquots of the supernatant were analyzed for Sr2`by atomic absorption spectroscopy. The procedure for determining the active uptake of Sr' involved comparing the net uptake in the presence and absence of substrate, as has previously been described (7).Mitochondria isolated from both plant and animal sources are capable of actively transporting divalent cations (1,5,9,10 RESULTSThe binding of small molecules to macromolecules can formally be described by equations analogous to those used to describe enzyme-substrate interactions (4). In plotting binding data Edsall and Wyman (4) have discussed the advantages of a Scatchard plot which formally coincides with the Eadie plot used in enzyme kinetics (3,16) and is based on the equation:Where V is the probability that a macromolecule chosen at random from a solution will have a molecule A attached to it, and K is the association constant for the interaction of the macromolecule P and A as follows:The symbol n is the number of binding sites at each macromolecule (4). In a plot of V/A against V when V/A = 0 then n = V, and the extrapolated intercept on the abscissa gives n, the maximum number of groups associated with binding. When