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K J Hellingwerf

Publications and source records attributed to K J Hellingwerf.

123 records · Page 7Linked to original sources
PubMed1980

Kinetic and steady-state investigations of solute accumulation in bacterial membranes by continuously monitoring the radioactivity in the effluent of flow-dialysis experiments.

The flow-dialysis technique for studies of solute accumulation by membrane preparations has been made more suitable for routine measurements by recording continuously the radioactivity in the effluent of a flow-dialysis vessel with a homogeneous flow-monitoring device for beta-emitters. This modification not only decreases the time and cost of a flow-dialysis experiment but also allows the investigator to react directly on the outcome of his experiments. Analysis of the kinetics of this automated flow-dialysis system shows that this technique can also be used for the determination of the rate of uptake of solutes into bacterial membranes. This has been confirmed in Rhodopseudomonas sphaeroides by comparing the results of Rb+ and inorganic phosphate uptake studies performed by automated flow-dialysis and by the conventional filtration procedure. This application has the limitation that solute uptake has to proceed linearly for a period of about five times the half-time of the response of the flow-dialysis system. The two described applications make automated flow-dialysis very well-suited for experiments on the bioenergetics and regulation of solute uptake into bacterial membranes. Both driving force and rate of solute uptake can now be determined in one experiment.

Cell Membrane↗
PubMed1979

Bacteriorhodopsin in liposomes. I. A description using irreversible thermodynamics.

A comprehensive description of light-induced ion transport in bacteriorhodopsin liposomes is presented. Linear irreversible thermodynamics and the chemiosmotic theory serve as theoretical bases for the formulation of a limited number of fundamental equations. In these equations mechanistic parameters characterize the dependence of ion movement and flux through the photochemical cycle of bacteriorhodopsin on electrochemical potential differences and a so-called light affinity. By making appropriate steady-state assummptions and carrying out mathematical reduction experimentally testable expressions, still containing the mechanistic parameters, are obtained. In the accompanying article rigid trials to falsify these expressions are shown to be unsuccessful.

Bacteriorhodopsins↗
PubMed1979

Bacteriorhodopsin in liposomes. II. Experimental evidence in support of a theoretical model.

In the preceding article equations describing relevant ion flows in illuminated suspensions of bacteriorhodopsin liposomes have been derived. Here these equations are subjected to experimental tests. Changes in permeability characteristics of the liposomal membrane are brought about by addition of specific ionophores and change of medium composition. Using light-driven proton uptake and electrochemical potential differences for protons across the membrane as observation parameters, ridig attempts to falsify the derived equations are unsuccessful. Agreement between equations and experimental results is established on the point of: (i) the antagonistic effect of valinomycin and nigericin on the two components of the proton-motive force, (ii) the time dependence of the changes in transmembrane electrical and chemical potential differences after the onset of illumination. In three independent experimental systems evidence was obtained for the correctness of the postulated dependence of the turnover rate of the photochemical cycle on back pressure by the transmembrane electrochemical potential difference for protons.

Bacteriorhodopsins↗
PubMed1978

Light-induced pH changes inside bacteriorhodopsin vesicles as measured by 31 P NMR.

31P NMR has been used to measure light-induced pH changes inside bacteriorhodopsin vesicles containing entrapped sodium glucose-6-phosphate. Reversible light-induced pH changes were observed at various pH values. The results indicate that our vesicle preparations were not homogeneous with respect to the generation of pH gradients.

Bacteriorhodopsins↗
PubMed1978

Bacteriorhodopsin vesicles. An outline of the requirements for light-dependent H+ pumping.

A systematic study was performed to determine under which conditions bacteriorhodopsin can be applied as an energy generator in reconstituted systems. It is concluded that reconstitution of an active light-driven proton pump is possible over a wide range of conditions. High extents (per bacteriorhodopsin molecule) of proton uptake by reconstituted vesicles are found at a high lipid to protein ratio, after long sonication and at high pH. No active proton pump is obtained if reconstitution is attempted at high pH with neutral phospholipids or at low ionic strength with negatively charged lipids. Attention was also paid to the requirement of a crystalline array for active pumping; most likely, monomeric bacteriorhodopsin molecules can effectively pump protons.

Bacteriorhodopsins↗
PubMed1975

Transport of amino acids in membrane vesicles of Rhodopseudomonas spheroides energized by respiratory and cyclic electron flow.

Active transport of amino acids in whole cells and membrane vesicles from the facultative photo-synthetic bacterium Rhodopseduomonas spheroides is coupled to electron flow in the respiratory chain and in the cyclic electron transfer system. In vesicles made from cells grown aerobically in the dark transport of amino acids is most effectively energized by the oxidation of NADH and to a lesser extent by ascorbate or succinate in the presence of N,N,N',N'-tetramethyl-1, 4-phenyldiamine dihydrochloride or by ascorbate + phenazine methosulphate via the respiratory chain with oxygen as terminal electron acceptor. In membrane vesicles from cells grown anaerobically in the light the energy for active transport of amino acids is supplied upon illumination by photo-oxidation of bacteriochlorophyll and subsequent electron flow through the cyclic electron transfer system. The initial rate of transport increases with the light intensity upto saturation levels. In both vesicle preparations, inhibitors of electron transfer flow inhibit amino acid uptake. In order to obtain light-driven amino acid transport the isolation of membrane vesicles needs to be performed in a medium with a redox potential between 0 and 100 mV. Illumination of vesicles made from cells grown anaerobically in the light results in the generation of a membrane potential as is indicated by the uptake of the lipophylic cation triphenyl-methylphosphonium.

Aerobiosis↗
PubMedDate not supplied

Rhodopsin(s) in eubacteria.

While the biochemical basis of photosynthesis by bacteriochlorophyll-based reaction centres in purple phototrophic Eubacteria and retinal-based bacteriorhodopsin in the Archaebacterium Halobacterium salinarium has been elucidated in great detail, much less is known about photosensory signal transduction; this is especially the case for Eubacteria. Recent findings on two different photosensory proteins in two different Eubacteria, which both show clear resemblances to the rhodopsins, will be presented. The photoactive yellow protein (PYP) from the purple phototrophic organism Ectothiorhodospira halophila probably functions as the photoreceptor for a new type of negative phototaxis response and has been studied in some detail with respect to its structural and photochemical characteristics. On basis of crystallographic an photochemical data it has been proposed that PYP contains retinal as a chromophore. However, we have unambiguously demonstrated that the PYP chromophore is different from retinal, in spite of the fact that PYP's photochemical properties show striking similarities with the rhodopsins. The cyanobacterium Calothrix sp. displays complementary chromatic adaptation, a process in which the pigment composition of the phycobilisomes is adjusted to the spectral characteristics of the incident light. In orange light the blueish chromophore phycocyanin is present, in green light the reddish phycoerythrin is synthesized. On the basis of the action spectrum of this adaptation process, we hypothesized that a rhodopsin is the photosensor in this process. In line with this, we found that nicotine, an inhibitor of the biosynthesis of beta-carotene (which is the precursor of retinal), abolishes chromatic adaptation. Direct proof of the involvement of a photosensory rhodopsin was obtained in experiments in which the chromatic adaptation response was restored by the addition of retinal to the cultures. The two photosensory proteins mentioned above represent the first examples of eubacterial photoreceptors that can be studied at a molecular level. Our current knowledge on these two proteins and their status as retinal proteins will be reviewed.

Journal Article↗
PubMedDate not supplied

(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system.

The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3- carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes.

Coumaric Acids↗