Yonosuke Kobatake

Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state.

SummaryThe membrane potential of mitochondria was estimated from the accumulation of tetraphenyl phosphonium (TPP+), which was determined with the TPP+-selective electrode developed in the present study. The preparation and some operational parameters of the electrode were described. The kinetics for uptake by mitochondria of TPP+ and DDA+ (dibenzyldimethyl ammonium) were analyzed, and it was found that TPP+ permeated the mitochondrial membrane about 15 times faster than DDA+. The final amounts of accumulation of TPP+ and DDA+ by mitochondria were approximately equal. For the state-4 mitochondria, the membrane potential was about 180 mV (interior negative). Simulataneous measurements of TPP+-uptake and oxygen consumption showed that the transition between states 3 and 4 was detectable by use of the TPP+-electrode. After the TPP+-electrode showed that state-4 was reached, the extramitochondrial phosphorylation potential was measured. The difference in pH across the membrane was measured from the distribution of permeant anion, acetate, so as to calculate the proton electrochemical potential. The ratio of extra-mitochondrial phosphorylation potential to proton electro-chemical potential,n was close to 3. This value ofn was also found to be 3 when ATP was hydrolyzed under the condition that the respiratory chain was arrested. The implication thatn=3 was discussed.

Selective electrode for dibenzyl dimethyl amoonium cation as indicator of the membrane potential in biological systems

The electrode sensitive to dibenzyl dimethyl ammonium (DDA+), which is considered to be an indicator of the membrane potential, was constructed by using tetraphenyl borone (TPB-) embedded in dichloroethane. Rapid and Nernstian responses were exhibited against DDA+ solutions ranging between 10(-2) and 3 - 10(-6) M in concentration. High selectivity for DDA+ was observed in the presence of various inorganis salts, ADP, ATP, oxidizable substrates and sugars. The electrode developed here was used to measure the DDA+ uptake in Streptococcus faecalis and the results agreed with those reported by Harold, F.M. and Papineau, D. ((1972) J. Membrane Biol. 8, 27-44 and 45-62). While they determined the DDA+ concentration in the medium by measuring the absorbance of the filtrate treated with the ion-exchangers, the electrode can measure directly the DDA+ concentration in the bacterial suspension without any any pretreatment. It was also shown that the electrode can measure the DDA+ uptake in mitochondria during energization.

Oscillating frequency of piezoelectric quartz crystal in solutions

Oscillating frequencies of a piezoelectric crystal were measured in various solutions. One side of the crystal surface was coated with a silicon sealant. This coating was useful for measuring the oscillation of crystals in solutions for a wide range of products of density (ϱ) and viscosity (η) and in electrolyte solutions. For measurement in solutions, the frequency change depended on the circuit used, whereas for measurements in air the circuit did not influence the frequency change. All experimental data showed that the frequency change from pure water, ΔFw, followed ΔFw = − K(√ϱη − √ϱwηw) except for electrolyte and polymer solutions, where K is a proportionality constant, ηw the density of pure water and ηw the viscosity of water.

A PVC-based electrode sensitive to DDA+ as a device for monitoring the membrane potential in biological systems.

Abstract A polyvinyl chloride (PVC)-based membrane electrode sensitive to dibenzyldimethyl ammonium cation (DDA+) was constructed, and operational parameters such as the selectivity coefficients, the detection limit, and the response time were obtained. In comparison with the selectivity coefficients obtained with the previous liquid-membrane electrode, significant improvement was not obtained, but the response time became pronouncedly shorter. Furthermore, the electrode lifetime was remarkably prolonged. With the electrode developed, the change in the membrane potential of liposomes containing dibutyl ferrocene which separated oxidizing and reducing agent solutions was measured. The DDA+ uptake, U, and the membrane potential estimated from U changed in accordance with the redox potential in the medium when the concentration of internal ferricyanide was kept constant. The membrane potential collapsed when the uncoupler of oxidative phosphorylation was added. The ANS fluorescence measurement indicated that negative charges appeared on energization with oxidizing-reducing agent. The change in membrane potential of mitochondria during energization was also measured. It was found that the liposome described above is a good model for the generation of membrane potentials in mitochondria.

Spatial and temporal organization of intracellular adenine nucleotides and cyclic nucleotides in relation to rhythmic motility in Physarum plasmodium.

Spatio-temporal organization of a migrating plasmodium was studied both by analysing intracellular concentrations of adenine and cyclic nucleotides and by applying image processing for recording oscillatory changes in thickness with use of microcomputers. ATP and ADP concentrations were about twice as high in the front as in the rear, while AMP distributed uniformly. On the other hand, cAMP and cGMP concentrations were several times higher in the rear than in the front, showing oscillations in between. The cAMP concentrations at the front oscillated with a phase advancing about one-third of the period with respect to the phase of the thickness oscillation, while cGMP concentration there varied only little. ATP concentration oscillated concomitantly with H+. A feedback control loop consisting of (ATP-H+)-cAMP-Ca2+ is proposed. The possible mechanism of rhythmic contractions involving mitochondria which may excrete pulses of Ca2+ and induce cell polarization is discussed.

Excitability, instability and phase transitions in squid axon membrane under internal perfusion with dilute salt solutions

Abstract Using giant axons of squid, Doryteuthis , available in Hokkaido, Japan, it was shown that axons internally perfused with a dilute sodium salt solution undergo an abrupt transition from a resting to a depolarized state on addition of KCl to an external medium containing CaCl 2 . Under internal perfusion with a dilute solution of sodium or cesium salt, it was possible to induce abrupt transitions between the two ( i.e. , resting and depolarized) states of the membrane by changing the temperature. “Giant fluctuations” in the state of the axon membrane were demonstrated at and near the critical points of the axon membrane. These findings are interpreted as supporting the view that an abrupt change in the membrane potential and conductance is an electrochemical manifestation of a phase transition of the membrane macromolecules.

Reversal of thermotaxis with oscillatory stimulation in the plasmodium of Physarum polycephalum

The plasmodium of Physarum polycephalum shows shuttle protoplasmic streaming and, upon steady local stimulation, moves toward or away from high or low temperatures, respectively. However, these thermotactic behaviors were reversed by oscillating the stimulus temperature. When the high temperature stimulus (originally an attractant) was oscillated slower than the intrinsic rhythm, the organism moved away from there as long as the entrainment was maintained. On the other hand, low temperature (a repellent), when oscillated faster to entrain the rhythm, attracted the organism. The implication of this result is discussed in relation to the mechanism of information processing in the plasmodium in terms of a coupled oscillator model.

Propagation of phase wave in relation to tactic responses by the plasmodium of Physarum polycephalum

The plasmodium of Physarum polycephalum shows two-dimensional patterns in thickness oscillation. Modulations of the oscillation pattern in response to local stimulation were studied by applying computer image processing. In a concentrically extending plasmodium, oscillations were entrained to a single frequency, and phase gradient vectors pointed radially and inward mostly. When a part of the plasmodium was exposed to different temperature or chemicals, the entrained oscillations continued but new propagating phase waves started within a few minutes around the stimulated region. Phase gradient vectors pointed away from attractive stimuli (high temperature, glucose, oatflakes) but toward repulsive ones (low temperature, salts, high osmolarity, anaerobic conditions). Thus, the sensed information seems integrated at the level of interacting oscillators.

Studies of Electric Capacitance of Membranes: I. A Model Membrane Composed of a Filter Paper and a Lipid Analogue

A hydrophobic filter paper of a given pore size containing a synthetic lipid, i.e. dioleyl phosphate, was interposed between aqueous electrolyte solutions having the same chemical composition and temperature. The electric capacitance and conductance of the membrane immersed in various concentrations of KCl were measured in the frequency range from 20 to 3 x 10(6) cycle/sec. The observed capacitance and conductance were found to be strongly dependent on the applied frequency. A theory is proposed to account for this dispersion of impedance observed in the present membrane-electrolyte system. The dispersion is attributed to the formation of bilayer membranes of the lipid inside the filter paper. The effects of the salt concentration, the adsorbed quantity of the lipid, and the pore size of the filter paper on the capacitance and conductance of the membrane are discussed in terms of the distribution function of bilayers formed within the filter paper.

Evidence that the long-lifetime photointermediate of s-rhodopsin is a receptor for negative phototaxis in halobacterium halobium

The effect of blue background light on behavioral response of Halobacterium halobium to step-like stimulation with green-orange attractant light was examined. The results strongly support the previously proposed hypothesis that a long-lifetime photointermediate of s-rhodopsin is the photoreceptor for repellent light: the step-like increase in green-orange light was convertible from attractant stimulus to repellent one, when the cells were constantly illuminated with blue light. No difference of the threshold intensity of the blue background light was observed between the mutant strain that lacks both bacteriorhodopsin and halorhodopsin and the wild type strain, suggesting that the two light-driven ion pumps are not participant in sensing attractant light.