Ion diffusion potentials across mycoplasma membranes determined by a novel method using a carbocyanine dye |
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Authors: | U Schummer H G Schiefer |
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Affiliation: | 1. Department of Oral Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan;2. Department of Orthodontics, Okayama University Hospital, Okayama 700-8558, Japan;3. Advanced Research Center for Oral and Craniofacial Sciences, Dental School, Okayama University, Okayama 700-8558, Japan;4. Division of Microbiology and Oral Infection, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Nagasaki University, Nagasaki 852-8588, Japan;5. Department of Molecular Microbiology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan;6. Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan |
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Abstract: | The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Electric membrane potential was calibrated vs fluorescence intensity of a potential-sensitive carbocyanine dye according to delta psi = (RT/F) X log([aIN(1 - IN) - b]/Kint), where IN = I/I0, I0 = maximal fluorescence intensity (obtained for delta psi----infinity), and a and b are constants. Fluorescence intensity was calibrated vs membrane potential by inducing a K+ diffusion potential. The calibration procedure was based on the assumption that in the presence of valinomycin the membrane potential was determined entirely by K+ diffusion. Then the dependence of fluorescence intensity on the external K+ concentration, Kext, could be described by Ival = I0[1 + a/(Kext + b)]-1. For Mycoplasma mycoides subsp. capri and enterococci, the constants were determined from experimental data using nonlinear least-squares computer-assisted methods. The validity of our assumption was proved using the "null-point" method. Here the Ca2+ ionophore A23187 and varying external Ca2+ concentrations were used to change the membrane potential experimentally. K+ and Na+ diffusion potentials significantly contributed to mycoplasma membrane potential whereas Cl- had no influence. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = -68 mV. |
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