Measurement of binding of adenine nucleotides and phosphate to cytosolic proteins in permeabilized rat-liver cells

1. A method is described for measuring the binding of metabolites to cytosolic proteins in situ in isolated rat-liver cells treated with filipin to render the plasma membrane permeable to compounds of low molecular weight. 2. There is no binding of ATP or inorganic phosphate to cytosolic proteins, either in the presence or in the absence of Mg2+. 3. Binding of ADP to cytosolic proteins occurs both in the absence and in the presence of Mg2+. The concentration of binding sites was 0.68 and 0.52 mumol/g dry weight of cells (n = 3-4) in the absence and presence of Mg2+, respectively. The corresponding Kd values were 320 microM and 235 microM