Reduction of membrane potential, an immediate effect of colicin K.

Abstract

(2), and inhibition of several active transport systems (3-6). Uptake and respiration of glucose remain normal, as do retention of a-methyl glucoside and facilitated diffusion of flD-galactosides (2).Previous experiments have shown that some of the observed changes are secondary effects (7). The processes that seem to be directly affected are those that are presumed to be dependent on an "energized membrane state." According to Mitchell's chemiosmotic hypothesis (8,9), the energized membrane state is identified as an electrochemical gradient of protons or protonmotive force (Ap), composed of a concentration gradient (ApH) and an electrical gradient (A41). The value of Ap is given in mV by the formula Ap = A4' -6OApH at 27°. Ap is postulated to be the driving force that energizes, either directly or indirectly, a wide variety of metabolic processes, including motility and various active transport systems. The primary effect of colicin K is apparently to uncouple the energy produced by respiration or by ATP hydrolysis from energy-utilizing processes such as active transport and motility. Colicin K might act either by lowering the energized membrane state or by preventing the utilization of the energized state. This paper presents evidence that colicin K acting on E. coil cells causes rapid membrane depolarization as measured by the accumulation of the lipophilic cation triphenylmethylphosphonium (TPMP+) (10). This conclusion is consistent with previous findings of Brewer (11) and Gould and Cramer (12). From measurements of the uptake of a weak acid analyzed by the flow dialysis method of Ramos et al. (13), we conclude that colicin K has only a slight effect on the pH gradient.In the course of these experiments we noticed that both the commonly used pretreatment of bacterial cells with EDTA and the presence of the lipophilic ions tetraphenylboron and TPMP+ have unexpected effects on certain bacterial membrane functions, effects that must be considered in the interpretation of experimental results. Ramos et al. (13). A pH electrode and a reference electrode (Radiometer G222C and K4112) were inserted in the cell suspension and connected to a recording pH meter. The flow rate of the dialysis buffer was about 5.1 ml/min. Fractions were collected directly into scintillation vials and sampled for radioactivity. The fraction collector and recorder were wired so that a small blip was recorded on the pH trace at the beginning of every fifth fraction.The method used by Ramos et al. (13) to calculate the amount of weak acid taken up and hence the internal pH had to be modified because our experiments, involving lower pH gradients and using dilute cell suspensions to maintain aerobiosis, required more precise measurements of relatively small changes in substrate concentration. A computer program was therefore devised § that calculates and plots the internal and external concentrations and the pH gradient. The same program was also used for proline uptake and can be used for any substrate that is actively concentrate...

Characterization of a membrane pore-forming protein from Entamoeba histolytica.

“…Among the bacterial poreforming proteins, perhaps most noteworthy are the colicins secreted by bacteria. Colicins El, Ia, and K depolarize E. coil (38)(39)(40), and colicin K is known to create voltage-dependent channels in the planar bilayer membrane (23). This change in ion permeability induced by colicins is compatible with a single-hit killing mechanism for target bacteria (23,33).…”

Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.

“…Colicin El is a 522-residue bactericidal protein, known to exert its lethal effect through an ability to depolarize the cytoplasmic membrane of sensitive Escherichia coli (1)(2)(3). Studies of colicin El action using artificial membranes have shown that the deenergization results from an ion channel formed by the colicin molecule, which dissipates the cellular membrane potential (ref.…”

Temperature‐dependent transmembrane potential changes in cells infected with a temperature‐sensitive moloney sarcoma virus

“…Transmembrane potential (Em) generally reflects the functional integrity and metabolic state of the cell. Alterations in Em are often the initial response to metabolic poisons (Weiss and Luria, 1978;Okada et al, 1980) or membrane modifiers such as lipids (Boonstra et al, 1982). Cone (1970) has proposed that Em is the regulator of mitosis in both normal and neoplastic cells, with depolarization initiating proliferation and polarization causing the cessation of growth.…”