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Biomedical subjects

R A Dilley

Publications and source records attributed to R A Dilley.

At least 55 records · Page 3Linked to original sources
PubMed1977

The effect of chloroplast coupling factor removal on thylakoid membrane ion permeability.

Removal of coupling factor protein (CF1) from spinach thylakoid membranes results in an enhancement of proton permeability but has no effect on chloride or potassium permeability. Anion permeability was measured by the rate of thylakoid packed volume changes. Potassium permeability was monitored by turbidity changes, packed thylakoid volume changes and ion flux studies using 86Rb+ as a tracer. 45Ca2+ was used to measure divalent cation fluxes. CF1-depleted chloroplasts had an unaltered rate of Ca2+ uptake, but the rate of Ca2+ efflux appeared to be increased. Calcium efflux rates could also be increased by the addition of a proton specific uncoupler, FCCP.

Calcium↗
PubMed1977

Chloroplast membrane sidedness. Location of plastocyanin determined by chemical modifiers.

Intact grana and stroma membranes (outer membrane absent) and detergent or sonication disrupted thylakoid membranes were treated with the hydrophilic covalent chemical modifiers [35S]diazonium benzene sulfonic acid ([35S]DABS) and [14C]glycine ethylester plus 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CDIS). Plastocyanin was purified using column chromatography followed by polyacrylamide gel electrophoresis and the incorporation of [35S]DABS and [14C]glycine ethylester into plastocyanin was determined by slicing the gels and counting the radioactivity in the plastocyanin band. Plastocyanin isolated from thylakoids disrupted prior to chemical modification binds two to four times as much of either modifier than the plastocyanin isolated from intact chloroplasts. This ratio is five to ten times lower than the ratio expected for a component buried behind the permeability barrier of a membrane. The data suggest that plastocyanin is partially exposed at the external surface of the thylakoid membrane rather than being completely buried in, or behind, the lipo-protein membrane.

Biological Assay↗
PubMed1976

Photophosphorylation as a function of illumination time. I. Effects of permeant cations and permeant anions.

(1) Very brief periods of illumination do not initiate photophosphorylation in isolated chloroplast lamellae. The time of illumination required before any phosphorylation can be detected is inversely proportional to the light intensity. At very high intensities, phosphorylation is initiated after illumination for about 4 ms. (2) There is no similar delay in the initiation of electron transport. The rate of electron transport is very high at first but declines at about the time the capacity for ATP synthesis develops. When the chloroplasts are uncoupled with gramicidin the high initial rate persists. (3) Various ions which permeate the thylakoid membrane (K+ or Rb+ in the presence of valinomycin, SCN-, I-, or C1O4-) markedly increase the time of illumination required to initiate phosphorylation. Potassium ions in the presence of valinomycin increase the delay to a maximum of about 50 ms whereas thiocyanate ions increase the delay to a maximum of about 25 ms. The effects of K+ with valinomycin and the effect of SCN- are not additive. Permeant ions and combinations of permeant ions have little or no effect on phosphorylation during continuous illumination. (4) The reason for the threshold in the light requirement and the reason for the effect of permeant ions thereon are both obscure. However, it could be argued that the energy for phosphorylation initially resides in an electric potential gradient which is abolished by migration of ions in the field, leaving a more slowly developing proton concentration gradient as the main driving force for phosphorylation during continuous illumination. If so, the threshold in the presence of permeant ions should depend on internal hydrogen ion buffering.

Adenosine Triphosphate↗
PubMed1976

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers.

(1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.

Adenosine Triphosphate↗
PubMed1976

Inhibition of energy-transducing functions of chloroplast membranes by lipophilic iron chelators.

Lipophilic metal chelators inhibit various energy-transducing functions of chloroplasts. The following observations were made 1. Photophosphorylation coupled to any known mode of electron transfer, i.e. whole-chain noncyclic, the partial noncyclic Photosystem I or Photosystem II reactions, or cyclic, is inhibited by several lipophilic chelators, but not by hydrophilic chelators. 2. The light- and dithioerythritol-dependent Mg2+-ATPase was also inhibited by the lipophilic chelators. 3. Electron transport through either partial reaction. Photosystem I or Photosystem II was not inhibited by lipophilic chelators. Whole-chain coupled electron transport was inhibited by bathophenanthroline, and the inhibition was not reversed by uncouplers. The diketone chelators diphenyl propanedione and nonanedione inhibited the coupled, whole-chain electron transport and the inhibition was reversed by uncouplers, a pattern typical of energy transfer inhibitors. The electron transport inhibition site is localized in the region of platoquinone leads to cytochrome f. This inhibition site is consistent with other recent work (Prince et al. (1975) FEBS Lett. 51, 108 and Malkin and Aparicio (1975) Biochem. Biophys. Res. Commun. 63, 1157) showing that a non-heme iron protein is present in chloroplasts having a redox potential near + 290 mV. A likely position for such a component to function in electron transport would be between plastoquinone and cytochrome f. just where our data suggests there to be a functional metalloprotein. 4. Some of the lipophilic chelators induce H+ leakiness in the chloroplast membrane, making interpretation of their phosphorylation inhibition difficult. However, 1-3 mM nonanedione does not induce significant H+ leakiness, while inhibiting ATP formation and the Mg2+-ATPase. Nonanedione, at those concentrations, causes a two- to four-fold increase in the extent of H+ uptake. 5. These results are consistent with, but do not prove, the involvement of a non-heme iron or a metalloprotein in chloroplast energy transduction.

Adenosine Triphosphatases↗
PubMed1975

The possible relationship between a membrane conformational change and photosystem II dependent hydrogen ion accumulation and adenosine 5'-triphosphate synthesis.

Data are presented which suggest that photosystem II dependent hydrogen ion accumulation and ATP synthesis can occur only after the lamellar membranes have undergone a conformational change. This membrane conformational change is detected by the electron transport dependent incorporation of diazonium benzene[35S]sulfonate into membrane components. Previously it was established that electron flux from the photosystem II primary acceptor to plastoquinone is a necessary event for the occurrence of the diazonium-detected conformational change. These studies indicate that the release of hydrogen ions during photosystem II oxidation of the primary reductant is also a necessary event for the diazonium-detected conformational change. When iodide were substituted for water (or other proton-releasing donors) as the primary reductant of system II the conformational change did not occur even though a substantial rate of electron flow from the primary acceptor to plastoquinone occurred.

Adenosine Triphosphate↗
PubMed1975

A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition.

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.

Benzenesulfonates↗
PubMed1974

Chloroplast grana membrane carboxyl groups: their involvement in membrane association.

Chloroplast membrane carboxyl groups were modified by carbodiimide activation followed by glycine methyl ester substitution, leaving the derivatized group uncharged. This charge alteration induced a number of effects similar to addition of salts to control chloroplasts suspended in a low salt medium. These include: (a) restacking or multiple membrane association in low salt-treated chloroplasts that lack grana stacks, (b) protection against polycation inhibition of photosystem I electron transfer, (c) reduction of the amount of polycations bound to the membranes, and (d) increased 90 degrees light scattering due to membrane conformational changes. Carboxyl modification also altered acid-induced conformational changes.These effects are interpretated as the results of the reduction in the surface negative charge contributed by carboxyl groups. Membrane structure at both a local, polyelectrolyte level and at the level of membrane-membrane interaction (stacking) is controlled in part by these negative, charged groups.

Journal Article↗
PubMed1972

Synthesis of plastoquinone analogs and inhibition of photosynthetic and mammalian enzyme systems.

New 5-hydroxy- and 5-chloro-6-alkyl-1,4-benzoquinones with one or two methyl groups on the nucleus were synthesized as potential antimetabolites of plastoquinones for biological research on photosynthetic and mammalian enzyme systems; the primary emphasis was on photosynthesis.2,3-Dimethyl-5-hydroxy-6-phytyl-1,4-benzoquinone completely inhibited in chloroplasts the water-dependent electron transport, but photosystem I was insensitive to this analog. The data are consistent with the interpretation that this analog inhibits electron transport in the chain prior to the site of electron donation from the ascorbate-dichlorophenolindophenol couple. Concentrations of 70 muM and 120 muM of this analog caused about 50 and 100% inhibition, respectively, of cyclic photophosphorylation.2,3-Dimethyl-5-hydroxy-6-phytyl-1,4-benzoquinone is a new type of inhibitor of photosynthetic electron transport that specifically inhibits the rate-limiting step between photosystems I and II. Structurally related analogs caused inhibitions in the range of 50-100% in chloroplasts. These analogs showed marginal inhibition in mitochondrial coenzyme Q(10)-oxidase systems from beef heart.

Animals↗