Effects of cytochalasin B on Na+ content and cell volume of Entamoeba histolytica

Cells of Entamoeba histolytica accumulated K+ and extruded Na+ compared to the concentrations of those ions present in the growth medium. Pinocytic activity, measured by the uptake of horseradish peroxidase of 125I-polyvinylpyrrolidone, was high (up to 0.3 ml/ml cells per h). Upon addition of cytochalasin B, at a concentration (20 microM) that completely blocked pinocytosis, cells lost up to 40% of their Na+ content within 90 min; K+ content was not affected or increased slightly compared to control cells without the inhibitor. Cation loss was associated with cell shrinkage. The dose-response curves for the effects of cytochalasin B on pinocytosis and Na+ content were identical. These data provide direct evidence that pinocytosis is an important component of the homeostatic system for Na+.     

Effects of amiloride on Na+ content and pinocytosis in Entamoeba histolytica

Amiloride, a blocker of Na+ leak and Na+-H+ exchange in animal cells, caused cells of Entamoeba histolytica to release Na+ (up to 40% of their original Na+ content within 90 min, at an amiloride concentration of 3 mM); K+ content was not affected. By comparing the unidirectional uptake of 22Na+ with that of the fluid-phase marker 125I-labeled poly(vinylpyrrolidone) we established that the amiloride-induced Na+ loss was due to inhibition of pinocytic Na+ uptake rather than to blockage of an amiloride-sensitive transport system in the plasma membrane. Amiloride penetrated the cells, and both its intracellular concentration and its effect on pinocytosis increased with pH. The permeant weak base quinacrine similarly inhibited pinocytosis in a pH-dependent manner. We conclude that the effect of amiloride on pinocytosis and, consequently, on Na+ content was due to its properties as a permeant weak base.     

The concept of chemical capacitance, A critique.

The concept of chemical capacitance as introduced by Hong and Mauzerall (Proc. Natl. Acad. Sci. U.S.A. 1974. 71:1564) is critically reexamined. This novel capacitance was introduced to explain the time-course of flash-induced photocurrents observed in lipid bilayer membranes containing porphyrins. According to Hong and Mauzerall, the chemical capacitance results from a combination of three fundamental capacitances: the geometric membrane capacitance and the two interfacial double layer capacitances. The concept of chemical capacitance is questioned for the following reasons: (i) The system analysis is insufficiently determinate. (ii) The measured chemical capacitance is approximately 0.16% of that predicted by the theory. (iii) The fact that only 20% of the membrane area is illuminated was not considered in the analysis. The latter point offers an alternative explanation of the capacitance in question: this capacitance may reflect that fraction of the total membrane capacitance that is photochemically active. If so, the concept of chemical capacitance lacks general significance.     

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680⁺ Phe^-) and charge stabilization (formation of P-680⁺ Q_A ^-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced Q_A was about 30% of that measured with oxidized Q_A. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNB m-dinitrobenzene - PPBQ phenyl-p-benzoquinone - PS I photosystem I of green plants - PS II photosystem II of green plants - PSU photosynthetic unit - P-680 primary donor of PS II - Phe intermediary pheophytin acceptor of PS II - Q_A primary quinone acceptor of PS II - RC reaction center     

I. Novel capacitative electrode with a wide frequency range for measurements of flash-induced changes of interface potential at the oil-water interface Mechanical construction and electrical characteristics of the electrode

The mechanical construction and the electrical properties of a new type of capacitative electrode for the oil-water interface are described. The electrode is designed to detect changes of the interface potential induced by photochemical, photophysical, and photobiological reactions occurring at the interface. The construction is based on capacitative coupling of two aqueous compartments separated by a thin Teflon film. Thereby, the oil-water interface is in horizontal position and the electrode is placed with its planar bottom about 10 μm above the interface. A main feature of the electrode is the transparency to visible light which is achieved by having a clear electrolyte solution in the inner compartment of the capacitative electrode.The aqueous subphase and the inner electrolyte are connected with AglAgCl electrodes to voltage amplifiers. The capacitative electrode is best operated under open circuit conditions. The frequency range experimentally verified is 500 $\text{MHz}\text{\$}\text{?}\text{?}{}_{\mathrm{<mtext>3</mtext><mtext>dB</mtext>}}\text{\$}\text{?}\text{0.03}\text{Hz}$. The sensitivity is mainly determined by the noise of the electronic amplifiers, typical 50–100 μV.     

Volume coding for BBA-Biomembranes

Photoconductivity changes in lipid bilayers containing magnesium-octaethylporphyrin were measured in KCl solutions. Light flashes increase the total current to at least twice the dark current under anaerobic conditions. Under aerobic conditions the photocurrent decreases to less than a third of the dark current and approaches zero as the oxygen concentration is increased. ESR measurements on liposomes containing magnesium-porphyrin are used to show that magnesium-octaethylporphyrin is converted to a cation, accompanied by oxygen consumption.     

Combined fluorescence and photovoltage studies on chlorosome containing bacteria

Energy transfer kinetics, primary charge separation, antenna size and excitonic connectivity of photosynthetic units (PSU) in whole cells of Chloroflexus aurantiacus were studied at room temperature by ps-fluorescence and ps-photovoltage as well as by stationary fluorescence-spectroscopy and fluorescence induction measurements. The fluorescence decay kinetics measured at different wavelengths are in accordance with the currently accepted sequential energy transfer from the chlorosomes via the baseplates to the B808–866 complexes and the final trapping in the RC with time constants of 19 ± 2 ps, 40 ± 4 ps and 90 ± 9 ps, respectively. However, the quantitative analysis of fluorescence spectra and the occurrence of slow phases in the fluorescence decays reveal that in whole cells a significant fraction of BChl c in the chlorosome and of BChl a in the baseplate is unconnected. The photovoltage kinetics consisted of two electrogenic phases with time constants of 118 ± 5 ps and 326 ± 35 ps and comparable electrogenicities. The first phase is ascribed to trapping from the B808-866 complexes by P+H_A- formation and the second one to charge stabilization on a quinone acceptor. Fluorescence induction curves displayed a pronounced sigmoidicity, indicating efficient lateral energy transfer between neighbored PSUs and a dense packing of 19 reaction centers (RC) beneath one chlorosome. A quantitative analysis of the fluorescence-induction curves at different excitation wavelengths allows the estimation of pigment stoichiometries (i.e. antenna sizes): BChl c/RC 794 and B808/RC 15.