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Biomedical subjects

J B Jackson

Publications and source records attributed to J B Jackson.

At least 163 records · Page 9Linked to original sources
PubMed1983

The influence of the ionic conductance on the relation between electron transport and proton-motive force in intact cells of Rhodopseudomonas capsulata.

1. The dependence of membrane potential (delta psi) on the rate of respiration in darkened intact cell suspensions of Rhodopseudomonas capsulata was distinctly non-linear: severe inhibition of respiration with either rotenone or KCN led to only a small drop in delta psi. 2. In the presence of 0.3 microMs carbonylcyanide p-trifluoromethoxyphenylhydrazone [CF3OPhzC(CN)2] the dependence of delta psi on respiratory rate became linear. Consequently, and particularly at lower concentrations of CF3OPhzC(CN)2, there was a pronounced, synergistic depression of the respiratory delta psi with CF3OPhzC(CN)2 and either rotenone or KCN. 3. Antimycin A, at a concentration which strongly inhibited the photosynthetic electron transport chain, only slightly lowered the light-induced delta psi in anaerobic cell suspensions. Antimycin and CF3OPhzC(CN)2 synergistically lowered delta psi generated by illumination. 4. The light-induced delta psi in anaerobic cells was only about 1.5-times larger than the respiratory-induced delta psi in darkened cells. Nevertheless it required approximately 16-times more CF3OPhzC(CN)2 to collapse the photosynthetic delta psi than the respiratory delta psi. 5. These results are discussed with reference to the ionic current/delta psi relation described in [J.B. Jackson (1982) FEBS Lett. 139, 139-143]. The unifying feature is that the intrinsic conductance of the cell membrane is strongly dependent on delta psi but the conductance due to CF3OPhzC(CN)2 is independent of delta psi.

Antimycin A↗
PubMed1983

Interaction between the respiratory and photosynthetic electron transport chains of intact cells of Rhodopseudomonas capsulata mediated by membrane potential.

1. The inhibition of respiration by light and the relief from this inhibition by uncoupling agents have been studied in intact cells of Rhodopseudomonas capsulata. 2. The same concentration range of carbonylcyanide p-trifluoromethoxyphenylhydrazone [CF3OPhzC(CN)2] prevented light-inhibition of respiration and depressed the membrane potential (delta psi) in illuminated, aerobic bacterial suspensions. 3. For a wide range of CF3OPhzC(CN)2 concentration the dependence of the respiration rate on delta psi in darkened cells coincided with that in illuminated cells. 4. When present separately, antimycin A and low concentrations of CF3OPhzC(CN)2 had little effect on light-inhibition of respiration or on delta psi in illuminated, aerobic cells, but added together they gave relief from light-inhibition and they lowered delta psi. 5. In the absence of CF3OPhzC(CN)2 even very low light intensities were sufficient to inhibit respiration and increase delta psi. In the presence of CF3OPhzC(CN)2 much higher light intensities were necessary. 6. The results are interpreted as evidence that light-inhibition of respiration is mediated by delta psi. The modification of the response by uncouplers, inhibitors and with light intensity is critically determined by the ionic conductance properties of the membrane in accordance with the findings in [Clark, A.J., Cotton, N.P.J., and Jackson, J.B. (1983) Eur. J. Biochem. 130, 575-580].

Antimycin A↗
PubMed1983

Electron flow to dimethylsulphoxide or trimethylamine-N-oxide generates a membrane potential in Rhodopseudomonas capsulata.

Under dark and essentially anaerobic conditions electron flow to either dimethylsulphoxide or trimethylamine-N-oxide in cells of Rhodopseudomonas capsulata has been shown to generate a membrane potential. This conclusion is based on the observation of a red shift in the carotenoid absorption band which is a well characterised indicator of membrane potential in this bacterium. The magnitude of the dimethylsulphoxide- or trimethylamine-N-oxide-dependent membrane potential was reduced either by a protonophore uncoupler of oxidative phosphorylation or synergistically by a combination of a protonophore plus rotenone, an inhibitor of electron flow from NADH dehydrogenase. These findings, together with the observation that venturicidin, an inhibitor of the proton translocating ATPase, did not reduce the membrane potential, show that electron flow to dimethylsulphoxide or trimethylamine-N-oxide is coupled to proton translocation. Thus contrary to some previous proposals dark and anaerobic growth of Rps. capsulata in the presence of dimethylsulphoxide or trimethylamine-N-oxide cannot be regarded as purely fermentative.

Anaerobiosis↗
PubMed1981

The measurement of membrane potential during photosynthesis and during respiration in intact cells of Rhodopseudomonas capsulata by both electrochromism and by permeant ion redistribution.

1. The membrane potential in intact cells of Rhodopseudomonas capsulata during photosynthesis and during dark respiration has been measured by two independent methods. 2. The light-induced and O2-induced shifts in the carotenoid absorption spectrum were measured in the intact cells. The shift was calibrated with K+-diffusion potentials in chromatophores derived from those cells. The light-induced and O2-induced membrane potentials were -290 mV and -230 mV respectively. 3. The energized uptake of butyltriphenylphosphonium ions was measured in the same batch of cells. The light-induced and O2-induced membrane potentials calculated from the Nernst equation were -160 mV and -120 mV respectively. 4. It is concluded that the two kinds of probe measure the electric potentials across different domains of the cytoplasmic membrane, but it is difficult to reconcile the existence of such domains with simple electrical analogues of the membrane and aqueous phases.

Bacterial Chromatophores↗
PubMed1981

Photosynthetic control and estimation of the optimal ATP: electron stoichiometry during flash activation of chromatophores from Rhodopseudomonas capsulata.

(1) When chromatophores from Rhodopseudomonas capsulata Ala pho+ are exposed to a train of high-frequency, saturating flashes the kinetics of the reaction centre bacteriochlorophyll absorption change enter a pseudo steady-state in which the extent of oxidation during the flashes is equal to the extent of reduction in between the flashes. The level of the pseudo steady-state is lowered by the presence of a phosphate acceptor system, raised by further addition of oligomycin, lowered by a combination of nigericin and valinomycin and raised by antimycin A. (2) In the pseudo steady-state, the extent of reaction centre bacteriochlorophyll oxidation taking place during the flash may be estimated by subtraction from the total concentration of reaction centre bacteriochlorophyll. This value is equated with the amount of electrons transported through the photosynthetic chain. Comparison with the measured ATP yield per flash in the pseudo steady-state permits calculation of the ATP: two electron ratio. The value of the ratio is 1.1 for flash frequencies between 3 and 12.5 Hz and declines at lower and higher frequencies. The ATP: two electron ratio is approximately halved in the presence of antimycin A. (3) An alternative estimate of the ATP: two electron ratio, based on the assumption that high-frequency flashes approximate to the condition of continuous illumination, was approx. 0.8.

Adenosine Triphosphate↗
PubMed1981

Circular dichroism, thermal denaturation, and deoxyribonuclease I digestion studies of nucleosomes highly enriched in high mobility group proteins HMG 1 and HMG 2.

Salt-soluble (S) nucleosomes that contain near equimolar high mobility group nonhistone chromosomal proteins HMG 1 and HMG 2 and lack histone H1 were isolated from mouse myeloma nuclei. Comparisons of the sedimentation, near-UV circular dichroism, thermal denaturation, and pattern of DNase I digestion of S nucleosomes with these properties of nucleosome cores or "typical" nucleosomes containing H1 did not detect significant differences. These results indicate that HMG 1 and 2 do not affect the conformation and stability of nucleosomes or nucleosomal DNA and are consistent with the proposal that major functions of HMG 1 and 2 are to replace H1 and maintain the (micro) solubility and accessibility of local chromatin regions. In contrast to these similarities, the initial rate of DNase I digestion of S nucleosomes was approximately 3 times that of chromatin depleted in S nucleosomes. This is consistent with a relation of S nucleosomes to transcription and suggests that subtle factors (not necessarily HMG 1 and 2) determine DNase I susceptibility.

Animals↗
PubMed1980

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata.

1. When cytochrome c2 is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction. 2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux. 3. At high redox potentials, where cytochrome c2 is unavailable for photooxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H+ efflux is nevertheless decreased by valinomycin/K+; demonstrating that the [BChl]2 leads to Q electron transfer generates a delocalised membrane potential.

Bacterial Chromatophores↗
PubMed1979

Correlation between ATP synthesis and the decay of the arotenoid band shift after single flash activation of chromatophores from Rhodopseudomonas capsulata.

ATP synthesis and the acceleration of the decay of the carotenoid absorption band shift after single flash excitation of Rhodopseudomonas capsulata chromatophores were compared. The two processes behave similarly with respect to: (1) ADP and Pi concentration; (2) inhibition by efrapeptin and venturicidin, and (3) inhibition by valinomycin/K+ and by ionophores. Taken together with earlier evidence for the electrochromic nature of the carotenoid band shift the data support the contention that positive charge moves outwards across the chromatophore membrane during ATP synthesis and justify the method for determination of the H+/ATP ratio (Petty, K.M. and Jackson, J.B. (1979) FEBS Lett. 97, 367-372). The ability of nucleotide diphosphates in the presence of Pi and Mg2+ to give rise to the acceleration of the carotenoid shift decay closely correlates with the rate of phosphorylation of the nucleotides in steady-state light. Nucleotide triphosphates enhance the decay in parallel with their rate of hydrolysis. Adenylyl imidodiphosphate is itself without effect on the decay of the carotenoid shift and it does not prevent the ADP-induced acceleration. The analogue does prevent the ATP effect but only after repeated flashes.

Adenosine Triphosphatases↗
PubMed1979

Kinetic factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation.

ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10-20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b/c2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.

Adenosine Triphosphate↗
PubMed1979

Chromatin fractionation procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins.

Initial results of an approach to the isolation of functionally active chromatin are described. Slight digestion of mouse myeloma nuclei at 0 degrees C with micrococcal nuclease, followed by dialysis against near-physiological saline solution containing 1 mM Mg2+, caused release of up to 17% of the nuclear DNA as soluble nucleoproteins. This soluble (S) fraction was relatively depleted in H1 histones and methylated DNA (5-methylcytosine) but highly enriched in RNA, single-stranded DNA, and nonhistone chromosomal proteins, particularly two species of the high mobility group identified as HMG 1 and HMG 2. The S fraction released most rapidly (6--8% of the total DNA) consisted mainly of mono- and small oligonucleosomes. The mononucleosomes appeared normal in terms of sedimentation behavior, DNA length, and content of histones H2A, H2B, H3, and H4, but lacked H1, and instead were associated with approximately stoichiometric amounts of HMG 1 and HMG 2. Studies using isolated, fluorescence-labeled, total mouse HMG proteins indicated that added HMG 1 and HMG 2 do not bind strongly to S-fraction nucleoproteins but that two smaller HMG species (probably HMG 14 and HMG 17) do bind preferentially to S-fraction mono- and dinucleosomes. These results argue against artifactual redistribution of HMG 1 and HMG 2 during this fractionation but suggest caution in interpreting the distribution of smaller HMG proteins after digestion of chromatin. The potential relationship of this soluble fraction to transcriptionally active chromatin is discussed.

Amino Acids↗
PubMed1979

Factors controlling the binding of two protons per electron transferred through the ubiquinone and cytochrome b/c2 segment of Rhodopseudomonas sphaeroides chromatophores.

1. On every turnover, 2.0 protons can be bound by the membrane for each single electron moving through the Q-b/c2 oxidoreductase. 2. One proton (H+II) binding reaction is, and one (H+I) is not, sensitive to antimycin. 3. The redox states of electron transfer components other than the proton binding agents can affect both the rate of proton uptake and the apparent pK values on the agents binding the protons. 4. The presence of valinomycin under certain well-defined conditions can strongly influence the value of the measured pK on the H+II binding agent.

Bacterial Chromatophores↗
PubMed1979

Transport of local anaesthetics across chromatophore membranes.

1. Both simple amines and tertiary amino local anaesthetics give rise to an accelerated decay of the absorption change of added pH indicator dyes and a decelerated decay of the endogenous carotenoid absorption band shift, following short flash excitation of Rhodopseudomonas sphaeroides chromatophores. 2. With increasing medium pH, lower concentrations of amine or local anaesthetics are effective. 3. The order of potency of the local anaesthetics concurs with their reported membrane/buffer partition coefficients and concentrations required for action potential blockade in nerve fibres. 4. The data are taken as evidence for rapid transport of the free base across the chromatophore membrane and relatively slow penetration of the protonated local anaesthetic. Protolytic reactions complete the effective dissipation of the trans-membrane pH gradient. 5. Benzocaine, with its unusually low pKa and the quaternary derivative, chloropromazine methiodide do not display this type of behaviour. 6. In the presence of membrane potential-collapsing agents, such as valinomycin/K+ or thiocyanate ions, local anaesthetics decelerate the decay of the cresol red change but have no effect on the carotenoid shift decay. It appears that transport of the unprotonated local anaesthetic although electrically neutral, requires the presence of a membrane potential. 7. In contrast, the non-anaesthetic amines act independently of the membrane potential. 8. Ca2+ interferes with the mechanism of local anaesthetic deceleration of the cresol red change decay in the presence of valinomycin/K+ or thiocyanate but not with other anaesthetic or amine reactions.

Bacterial Chromatophores↗
PubMed1978

Affinity chromatography of H+-translocating adenosine triphosphatase isolated by chloroform extraction of Rhodospirillum rubrum chromatophores. Modification of binding affinity by divalent cations and activating anions.

1. ATPase isolated from Rhodospirillum rubrum by chloroform extraction and purified by gel filtration or affinity chromatography shows three bands (alpha, beta and gamma) upon electrophoresis in sodium dodecyl sulphate. 2. Ca2+-ATPase activity of the preparation is inhibited by aurovertin and efrapeptin but not by oligomycin. Activity may be inhibited by treatment with 4-chloro-7-nitrobenzofurazan and subsequently restored by dithiothreitol. 3. The enzyme fails to reconstitute photophosphorylation in chromatophores depleted of ATPase by sonic irradiation. 4. Most of the active protein from the crude chloroform extract binds to an affinity chromatography column bearing an immobilised ADP analogue but not to a column bearing immobilised pyrophosphate. 5. In the absence of divalent cations, a component with a very high specific activity for Ca2+-ATPase is eluted from the column by 1.6 mM ATP. This protein migrates asa single band on 5% polyacrylamide gel electrophoresis and only possesses three subunits. At 12 mM ATP an inactive protein is eluted which does not run on acid or alkali polyacrylamide gels and shows a complex subunit structure. 6. ATPase preparations prepared by acetone extraction or by sonic irradiation of chromatophores may also be purified 10-fold by affinity chromatography. 7. The inclusion of 5 mM MgCl2 or CaCl2 during affinity chromatography of chloroform ATPase increases the capacity of the column for the enzyme and demands a higher eluting concentration of ATP. 8. When the enzyme is more than 90% inhibited by efrapeptin or 4-chloro-7-nitrobenzofurazan, the binding characteristics of the enzyme are not affected. 9. 10 mM Na2SO3, which greatly stimulates the Ca2+- and Mg2+-dependent ATPase activity of the enzyme and increases Ki (ADP) for Ca2+-ATPase from 50 to 850 micron, prevents binding to the affinity column. Binding may be restored by the addition of divalent cations. 10. Na2SO3 increases the rate of ATP hydrolysis, ATP-driven H+ translocation and ATP-driven transhydrogenase in chromatophores. 11. It is proposed that anions such as sulphite convert the chromatophore ATPase into a form which is a more efficient energy transducer.

Adenosine Triphosphatases↗