Ion diffusion potentials across mycoplasma membranes determined by a novel method using a carbocyanine dye.
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.
D052160:Benzothiazoles; D002118:Calcium / Q000378:metabolism; D002232:Carbocyanines; D002712:Chlorides / Q000378:metabolism; D004058:Diffusion; D005453:Fluorescence; D008564:Membrane Potentials; D009174:Mycoplasma / Q000502:physiology; D011188:Potassium / Q000378:metabolism; D011804:Quinolines; D012964:Sodium / Q000378:metabolism
D052160:Benzothiazoles; D002232:Carbocyanines; D002712:Chlorides; D011804:Quinolines; C012944:3,3'-dipropyl-2,2'-thiadicarbocyanine; D012964:Sodium; D011188:Potassium; D002118:Calcium