Bacterial cytoplasmic membrane permeability assay using ion-selective electrodes.
We used K(+) and tetraphenylphosphonium (TPP(+)) electrodes simultaneously to evaluate the ability of antimicrobial peptides to form channels (or more generally to increase permeability) and to abolish membrane potential in bacterial cytoplasmic membranes in situ. Such evaluations are usually made independently by colorimetric monitoring of the hydrolysis of a chromogenic substrate by a cytoplasmic enzyme or by fluorimetric determination of membrane depolarization using a membrane potential-sensitive dye. In the present study, the K(+) electrode was used to evaluate channel-forming ability by monitoring the efflux of K(+) originally present in the cytoplasm of bacteria, while the TPP(+) electrode was used to examine membrane depolarization causing the efflux of TPP(+) accumulated in the cytoplasm of bacteria dependent on membrane potential. Thus, the combination of these two electrodes enabled us to clarify how the peptide-induced formation of ion channels is involved in disrupting the energy-generating system in situ.
Ohmizo
Chie
C
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.
Yata
Masaya
M
Katsu
Takashi
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D017736:Ion-Selective Electrodes; D008564:Membrane Potentials / Q000502:physiology; D008826:Microbial Sensitivity Tests; D009861:Onium Compounds / Q000378:metabolism / Q000493:pharmacokinetics; D009943:Organophosphorus Compounds / Q000378:metabolism / Q000493:pharmacokinetics; D010455:Peptides; D011188:Potassium / Q000378:metabolism / Q000493:pharmacokinetics; D013211:Staphylococcus aureus / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure
D000900:Anti-Bacterial Agents; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D010455:Peptides; D011188:Potassium; C013289:tetraphenylphosphonium