Interaction of the Complex between Cloacin and Its Immunity Protein and of Cloacin with the Outer and Cytoplasmic Membranes of Sensitive Cells

1 The interaction of a complex of [14C]cloacin and its immunity protein (cloacin complex) and of [14C]cloacin itself with the outer and cytoplasmic membranes of sensitive cells has been studied to elucidate the difference in specific killing activity of both bacteriocin preparations [Eur. J. Biochem. 73, 107–114 (1977)]. 2 No significant difference exists between cloacin complex and cloacin with respect to the specificity of adsorption, the adsorption characteristics and the kinetics of binding to the outer membrane receptors of sensitive cells grown in brain/heart infusion (about 425 receptors per cell). 3 The adsorption of the bacteriocin onto the receptors shows a temperature optimum between 35°C and 40°C, but is not affected by variation in pH between 5.0 and 8.0 or extracellular potassium chloride up to a concentration of 0.5 M. An apparent association constant of 0.6 × 108 M−1 is calculated at 37°C and pH 7.0. 4 At 37°C and pH 7.0 virtually all the cloacin molecules adsorbed by sensitive cells can be digested by trypsin during at least 3 h after adsorption, in contrast to the adsorbed molecules of cloacin complex which become gradually less accessible to trypsin. 5 No significant difference exists between the affinity of cloacin complex and cloacin for cytoplasmic membrane vesicles isolated from sensitive cells. At 37°C and pH 7.0 about 105 bacteriocin molecules can bind per vesicle with an apparent association constant of about 8 × 108 M−1. 6 These results indicate that most probably the removal of immunity protein from the cloacin complex in vitro results in a strong reduction of the transfer of biologically active cloacin molecules from the cell surface receptors to the cytoplasmic membrane in vivo. 7 This conclusion is confirmed by the observation that, in vivo, cloacin itself is less effective in the induction of a leakage of potassium ions across the cytoplasmic membrane compared to the cloacin complex. Concomitant with the reduction of the leakage of potassium ions a decrease in killing activity is observed. Increasing concentrations of extracellular potassium chloride gradually reduce the killing activity of cloacin complex to 3.1% and of cloacin to 45.5% in the presence of 0.2 M potassium chloride.