L. Keszthelyi

Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis

The kinetics of the photoreceptor potential of phototaxis in biflagellated green alga Haematococcus pluvialis in response to a 10-ns laser pulse of three wavelengths (465, 550, and 590 nm) were measured in single cells with 30 mus time resolution. The rise and the decay of photoinduced potential are both at least biphasic. The first component of the rise is very stable and has no measurable (<30 mus) time delay. The second component is triggered after a 120-400-mus lag period, depending on flash intensity. Its appearance is sensitive to the physiological state of the cell and the amplitude can be increased by phototactically ineffective red background illumination. The electrical generators for both components are localized in the same region of the cell membrane (on the stigma-bearing side) and these components have the same depolarizing sign. The results indicate that the photoreceptor potential in phototaxis comprises two components, which could be interpreted as light-induced charge movement within the photoreceptor molecules and changes in ion permeability of the cell membrane.

Photoelectric responses in phototactic flagellated algae measured in cell suspension

Abstract A new method for the investigation of electric responses involved in the light reception in microorganisms has been developed. It is based on the detection of photoelectric signals in suspensions of cells (instead of a single cell) by two different techniques: (a) by unilateral excitation of non-oriented cells and (b) after preorientation of the cells (e.g. by gravitaxis or weak, phototactically active light). The method was applied to the flagellated green algae Haematococcus and Chlamydomonas (and several of its mutants). Three main components of the electric signal, which differs in their origin and the mechanisms underlying that registration, were identified. Fast (microsecond) responses reflect charge separation in reaction centres of photosynthesis and are due to the classical light gradient effect on unilateral flash excitation. The later components of the electric signal are involved in photoreception and represent the photoreceptor potential of phototaxis and the calcium-dependent regenerative response. They are measured because of the directional sensitivity of the photoreceptor antenna and the asymmetry of localization of the electric currents involved in the sensory transduction chain. A general similarity between the electric responses in both organisms shows that the sensory transduction chain of photomovements in Chlamydomonas is similar to that described previously for Haematococcus. The advantages of the proposed method are discussed.

Electro-optical measurements on aqueous suspension of purple membrane from Halobacterium halobium.

The permanent dipole moment, polarizability, and the retinal angle of Halobacterium halobium purple membranes were determined at different pH values. All of the parameters have a maximum between pH 5 and 6. There is a reversal in the direction of the permanent dipole moment near pH 5. The value of permanent dipole moment was determined to be 60 D/protein at pH 6.6, and the value obtained for polarizability was 3 X 10(-28) Fm2/membrane fragment. The retinal angle of all-trans retinal was 0.8 degrees smaller than that of the 13-cis conformation.

Electric response of a back photoreaction in the bacteriorhodopsin photocycle

The electric response of a back photoreaction in the bacteriorhodopsin photocycle was investigated. The proton pumping activity of green flash excited bacteriorhodopsin stops if the M412 form is illuminated by blue light (Karvaly and Dancsházy, 1977). In the present work a fast negative displacement current signal was measured in an oriented membrane suspension system, indicative of back movement of protons from M412 to BR570. Quantitative evaluation of the data shows that there are at least two steps in the back reaction, with different rate constants. The temperature dependence of the rate constants show simple linear Arrhenius behavior between 5 degree and 40 degree C. The rate constants were slower by a factor of 1.8 in D2O suspension. The relevance of the protein electric response signals (PERS) observed in this paper to the early receptor potential is discussed.

Protein electric response signals from dielectrically polarized systems

Charges move, dipoles rotate inside the proteins during the functioning. A few examples: in myoglobin the Fe z+ ion moves in and out the heme plane during binding and release of CO (Phillips, 1978); charged particles or dipoles move when Na+-K + channels are opened and closed (gating current; Armstrong & Gully, 1979); and charged particles move through proteins in case of ion pumps (for example, Na +,K+-ATPase translocates Na + and K + ions; Kyte, 1981). Moving charges, rotating dipoles in proteins (as in any dielectric medium), induce displacement currents. With suitable methods the kinetics of these currents can be measured, providing important information about the processes related to the activity of proteins. This electrical signal can be named protein electric response signal (PERS) (Keszthelyi & Ormos, 1980). Two requirements have to be fulfilled in order to observe the time course of PERS: (i) a large number of proteins should be synchronized in their function, and (ii) the systems must be asymmetric with respect to the measuring electrodes. The first requirement can be easily met in the case of light-activated systems by short light-flash excitation. Most systems can be synchronized by light-flash excitation in an indirect way by the lightinduced release of protons (Gutman, 1986) and ATP (Christensen et al., 1988), Ca, etc., from caged substances. Several systems can be excited with electric pulses (as in the case of neurons). The second requirement needs asymmetric systems located between two electrodes. Membranebound proteins are the first candidates if (i) they are in the outer membrane of large enough cells, and the two electrodes can be located inside the cell and

Bacteriorhodopsin as a possible chloride pump

Purple membranes oriented and immobilized in gel show charge transfer at pH 0.55 if the pH is set by HCl. Current appears as laser flash driven transient and also as continuous current by quasi‐continuous illumination. If the pH value 0.55 is set by H2SO4 continuous current is not observed. The results suggest that bacteriorhodopsin may pump chloride ions at low pH.

Charge Motion during the Photocycle of Bacteriorhodopsin

The function of bacteriorhodopsin in Halobacterium salinarum is to pump protons from the internal side of the plasma membrane to the external after light excitation, thereby building up electrochemical energy. This energy is transduced into biological energy forms. This review deals with one of the methods elaborated for recording the charge transfer inside the protein. In this method the current produced in oriented purple membrane containing bacteriorhodopsin is measured. It is shown that this method might be applied not only to correlate charge motion with the photocycle reactions but also for general problems like effect of water, electric field, and different ions and buffers for the functioning of proteins.

The effect of pH on proton transport by bacteriorhodopsin

Abstract The pH-dependence of proton motion during the photocycle was investigated by measuring the photoelectric signals due to charge displacement inside bacteriorhodopsin molecules. Measurements were performed on purple membranes oriented in suspension and the kinetics of flash excited electric and light absorption signals was compared. It was found that in the pH range 4.5–8 the photocycle and the successive proton movements have identical kinetics, and do not depend on pH. In the pH range 8–10 both kinetics change, though differently; the charge motion decouples from the photocycle and the photocycle seems to split up into two parallel paths, the photoelectric signal becomes faster. However, the net proton transfer remains the same as at lower pH values. Above pH ≈ 10, the photocycle behaves differently and cannot be described by the parallel pathway model and the net proton displacement drops. The results are explained by the successive titration of two groups (probably tyrosine) participating in proton translocation.

Diamines reverse the direction on the bacteriorhodopsin proton pump

Purple membranes oriented and immobilized in gel were soaked in solutions containing different monoamines and diamines. The flash‐excited electric signals showed a reversal of the direction of the charge motion in bacteriorhodopsin in the case of diamines which was interpreted as the reversal of the proton pump. Monoamines had negligible influence on the electric signals. The influence of tetramethylethylenediamine was also studied in detail.

Electrooptical measurements on purple membrane containing bacteriorhodopsin mutants.

Electrooptical measurements on purple membrane containing the wild-type and 10 different bacteriorhodopsin mutants have shown that the direction of the permanent electric dipole moment of all these membranes reverses at different pH values in the range 3.2-6.4. The induced dipole moment and the retinal angle exhibit an increased value at these pHs. The results demonstrate that the bacteriorhodopsin protein makes an important contribution to the electrooptical properties of the purple membrane.