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Biomedical subjects

A R Crofts

Publications and source records attributed to A R Crofts.

At least 73 records · Page 4Linked to original sources
PubMed1980

The photosynthetic electron transfer chain of Chromatium vinosum chromatophores: flash-induced cytochrome b reduction.

Reduction of a cytochrome b following excitation by a single, short, near-saturating light flash has been demonstrated in Chromatium vinosum chromatophores. The extent of reduction is increased by addition of antimycin. The cytochrome has an alpha-band maximum at 562 nm in the presence of antimycin. The cytochrome b reduction is most readily observed in the presence of antimycin at high redox potential when cytochrome c-555 is oxidised before excitation. Under these conditions the half-time for reduction is about 20 ms, and the extent is about 0.5 mol of cytochrome b reduced per mol of reaction center oxidised. This extent of reduction is observed on the first flash-excitation from the dark-adapted state, and there was no indication that the reaction center quinone acceptor complex acted as a two-electron accumulating system. With cytochrome c-555 reduced before excitation, the extent of cytochrome b reduction is approximately halved. The factors which result in substoichiometric cytochrome b reduction are not yet understood. Agents which appear to inhibit primary acceptor oxidation by the secondary acceptor (UHDBT, PHDBT, DDAQQ, HOQNO, o-phenanthroline), inhibit reduction of the cytochrome b. DBMIB inhibits cytochrome b reduction but does not appear to inhibit primary acceptor oxidation. These observations confirm that a cytochrome b receives electrons delivered from the primary acceptor complex, and indicate that the photoreduced cytochrome b is reoxidised via an antimycin-sensitive pathway.

Antimycin A↗
PubMed1980

Binary oscillations in the rate of reoxidation of the primary acceptor of photosystem II.

The decay of fluorescence yield following each of a series of saturating laser flashes has been used to monitor the kinetics of reoxidation of the primary acceptor of Photosystem II under conditions of varied redox potential. 1. In dark-adapted chloroplasts, a damped binary oscillation as a function of flash number was observed in the kinetics of the decay of the fluorescence yield. The decay was faster on odd than on even-numbered flashes. 2. In the presence of low concentrations of 1,4-benzoquinone, the oscillation was more marked, and over the range approx 200--350 mV, independent of redox potential. The decay following flash 1 under these conditions had a half-time of approx. 200--400 microseconds. The decay following flash 2 was decelerated; the initial rate was up to 10-fold slower than after flash 1. 3. We suggest that the kinetics following a single flash reflect the rate of the reaction Q-B leads to QB-, and following the second flash, Q-B- leads to QB2-. Benzoquinone at low concentrations oxidises a residual fraction of B- which is usually reduced in the dark before the flash sequence. 4. A faster component in the decay (t u/2 approximately 140 microseconds) following the first flash titrated in over the range Eh greater than 350 mV. The binary oscillation was still apparent but delayed by one flash. 5. We discuss the relative redox potentials of the couples B/B- and B-/B2-, and the role of the component which titrates in at Eh greater than 350 mV.

Chloroplasts↗
PubMed1980

The quantum efficiency of proton pumping by the purple membrane of Halobacterium halobium.

The quantum yield of H+ release in purple membrane (PM) sheets, and H+ uptake in phospholipid (egg phosphatidylcholine, PC) vesicles containing PM, was measured in single turnover light flashes using a pH-sensitive dye, p-nitrophenol, with rhodopsin as an actinometer. We have also calculated the ratio of H+ released per M412 formed (an unprotonated Shiff-base intermediate formed during the photocycle). In PM sheets, the quantum yield of H+ release depends on the medium. The quantum yield of M412 is independent of salt concentration. The ratio H+/M412 is approximately 1.8 M KC; and approximately 0.64 in 10 mM KCl. Direct measurements of the quantum yield of H+ give approximately 0.7 when the PM is suspended in 0.5 M KC; and 0.25 in 10 mM KCl. Using a quantum yield for M412 formation of 0.3 (Becher and Ebrey, 1977 Biophys J. 17:185.), these measurements also give a H+/M412 approximately 2 at high salt. In PM/PC vesicles, the H+/M412 is approximately 2 at all salt concentrations. The M412 decay is biphasic and the dye absorption change is monophasic. The dissipation of the proton gradient is very slow, taking on the order of seconds. Addition of nigericin (H+/K+ antiporter) drastically reduces the pH changes observed in PM/PC vesicles. This and the observation that the proton relaxation time is much longer than the photochemical cycling time suggest that the protons are pumped across the membrane and there is no contribution as a result of reversible binding and release of protons on just one side of the membrane.

Bacteriorhodopsins↗
PubMed1979

A high potential acceptor for photosystem II.

The effects of ferricyanide on Photosystem II reactions have been investigated by measurements of microsecond and millisecond prompt fluorescence and microsecond-delayed fluorescence in dark-adapted chloroplasts: (1) Titrations using ferri-ferrocyanide mixtures on: (a) the fast phase of the increase in fluorescence yield observed during a xenon flash, and (b) the normalised area above the millisecond fluorescence induction curve for chloroplasts inhibited by DCMU, showed a pH dependent mid point potential of 400 mV at pH 7.0 which varied by approx. -60 mV/pH unit between pH 6 and 8.5. (2) A saturating laser flash induced a fluorescence increase (as monitored by a weak measuring beam) of only 50% of that reached following a second flash in chloroplasts preincubated with ferricyanide and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prior to illumination. In the absence of ferricyanide, the fluorescence level reached after a single flash was initially close to that measured after a second flash (although the level subsequently declined). (3) The initial amplitude of the microsecond-delayed fluorescence excited by a single laser flash was diminished in chloroplasts dark-adapted with ferricyanide. In the presence of DCMU and ferricyanide, the amplitude was also diminished for the first flash of a series, but subsequently enhanced above the level obtained in chloroplasts in the presence of DCMU alone. (4) The above effects were not seen if DCMU was added to the chloroplasts before ferricyanide, or if the period of incubation with ferricyanide was much less than 4 min. (5) These results suggested the presence of a second acceptor Q2, with Em7 = 400 mV and n = 1, before the DCMU block in Photosystem II. There is 0.35--1 equivalent of the acceptor per reaction centre, and its reduction occurs within less than 5 mus. The role of the acceptor in double turnovers of the photochemistry during a single flash and its likely operating redox potential are discussed.

Chloroplasts↗
PubMed1979

Effects of pH on reactions on the donor side of photosystem II.

The effects of pH on the increase of fluorescence yield measured in the microsecond range, and on the microsecond delayed fluorescence have been studied in dark adapted chloroplasts as a function of flash number. (1) At pH 7, the amplitude of the fast-phase of the microsecond fluorescence yield rise oscillated as a function of flash number with period 4 and with maxima on flashes 1 and 5, and minima on flashes 3 and 7. The damped oscillations were apparent over the range between 6 and 8, although the absolute amplitude of the fast phase was diminished at the lower end of the range. At pH 4, there was no fast phase in the rise and, at pH 9, an enhanced fast-phase occurred only for the first flash. (2) The decay of microsecond delayed fluorescence was described by the sum of exponentials with half-times of 10--15 mus and 40--50 mus. Over the pH range 6- less than 8, the extrapolated initial amplitude and the proportion of the change due to the faster component showed oscillations which were opposite in phase to those observed for the prompt fluorescence yield rise; the slower component showed weaker oscillations of the same phase. At pH 4, there were no oscillations and the slow phase predominated. At pH 9, the delayed fluorescence intensity was diminished on the first flash, and high on subsequent flashes. (3) The results are interpreted in terms of a model in which protons are released during all transitions of the S-states with the exception of S1 leads to S2, and in which ther are two sites of inhibition on the donor side of the photo-system at extreme pH values. At pH 4, electron donation to P+ occurs with a half-time approx. 135 mus, either by a back reaction from Q-, or from D; electron transport is interrupted between Z1 and P. At pH 9, electron transport is inhibited between Z1 and Z2; rapid re-reduction of P+ by Z1 occurs after 1 flash, and on subsequent flashes electrons from D, an alternative donor reduce P+. The location of the positive charge on states S2 and S3 is discussed.

Chloroplasts↗
PubMed1978

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides.

The pigment content of a B800-850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed. The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800-850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band. The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.

Bacterial Chromatophores↗
PubMed1978

The initial stages of photophosphorylation. Studies using excitation by saturating, short flashes of light.

1. Photophosphorylation was studied in spinach chloroplasts on illumination, from the dark state, with saturating short ("single turnover") flashes of light. 2. At rapid flash rates (100 Hz), phosphorylation began within the first five flashes. The ATPase inhibitor protein appeared to be displaced from its inhibitory site on the ATPase also within five flashes, as deduced from the flash-induced ATPase activity. 3. At slower flash rates, or if the rate of electron transfer were reduced with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), phosphorylation began only after a larger number (50--60) of flashes. The displacement of the ATPase inhibitior protein was similarly delayed. 4. Partial displacement of the inhibitor protein from its inhibitory site on the ATPase (by pretreatment with dithioerythritol) allowed phosphorylation to proceed without a perceptible lag, even in the presence of DCMU. It was concluded that the ATPase inhibitor protein must be displaced on the ATPase before phosphorylation can begin, and that this process is energy dependent. 5. During the flash regime used, release of inhibitor from its inhibitory site seemed to be governed largely by the membrane potential. The light-induced pH gradient seemed to have little effect under these conditions. Our results are not compatible with a direct conformational interaction between the electron transfer chain and the ATPase causing displacement of the inhibitor. 6. The maximal rate of photophosphorylation induced by less than 200 flashes was 0.12--0.15 mol ATP made/mol ATPase per flash. This rate seemed to be limited not be the supply of energy to the ATPase molecules, nor by the maximal turnover capacity of the ATP synthesising system, but by the number of ATPase molecules which were active in synthesis, i.e., which lacked the inhibitor protein. 7. The bound nucleotides of the coupling ATPase exchanged with added nucleotides during single turnover flashes. At high flash rates, exchange began within 5 flashes. The average amount of nucleotide exchanged per flash over 100 flashes was about one tenth the amount of ATP synthesised in each flash. 8. It is concluded that, during phosphorylation, a steady state level of active coupling ATPases is set up. The energy-dependent displacement of the inhibitor protein, and its (energy-independent) relaxation back on to the inhibitory site are the two opposing factors involved in this steady state.

Adenosine Triphosphatases↗
PubMed1977

The carotenoid shift in Rhodopseudomonas sphaeroides. Change induced under continuous illumination.

The spectrum of the carotenoid shift generated under continuous illumination in the GIC mutant of Rhodopseudomonas sphaeroides, which has a single carotenoid, has been examined under a variety of conditions expected to alter the size of the membrane potential. If the difference spectrum observed was due to a species with the spectrum of the bulk pigment, it would correspond to a change of a variable proportion of the pigment to a form absorbing at a higher wavelength. The maximal change induced by light could be described as a shift of about 10% of the pigment by 7 nm to the red, assuming that the shifted species was spectrally identical to the bulk carotenoid. It is concluded that the changes seen are not easily compatible with a progressive red shift in the whole spectrum with increasing applied potential as would be expected from a simple linear electrochromic mechanism; alternative hypotheses are discussed.

Bacterial Chromatophores↗
PubMed1977

The carotenoid shift in Rhodopseudomonas sphaeroides. The flash induced change.

A mutant, Rhodopseudomonas sphaeroides GIC, having only one major carotenoid, neurosporene, is described. The spectrum of the carotenoid shift in this mutant is analysed and it is concluded that only 7-11% of the pigment is involved under conditions of steady-state illumination and that this pigment undergoes a shift of 7 nm. The spectrum of the carotenoid shift under conditions of multi-flash illumination is examined for changes in shape concordant with a progressive red shift of the pigment with increasing membrane potential; the spectra of the fast change after each of three flashes does not agree well with predictions from a model involving a progressive shift of the pigment, the slow change shows qualitative agreement with such a model but the small size of the signal and the presence of more than one phase makes analysis of this phase more difficult. No separate pool of carotenoid, that might correspond to that postulated to participate in the carotenoid shift, could be identified by fourth derivative analysis of, or curve fitting to, the spectrum of the neurosporene.

Antimycin A↗
PubMed1975

Asymmetry of an energy transducing membrane the location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata.

Monospecific antibodies have been prepared against cytochrome c2 from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c' from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth. Cytochrome c2 is located in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.

Animals↗