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R E McCarty

Publications and source records attributed to R E McCarty.

At least 73 records · Page 4Linked to original sources
PubMed1981

Quantitative aspects of adenosine triphosphate-driven proton translocation in spinach chloroplast thylakoids.

After illumination in the presence of dithiothreitol, chloroplast thylakoids catalyze ATP hydrolysis and an exchange between ATP and Pi in the dark. ATP hydrolysis is linked to inward proton translocation. The relationships between ATP hydrolysis, ATP-Pi exchange, and proton translocation during the steady state were examined. The internal proton concentration was found to be proportional to the rate of ATP hydrolysis when these parameters were varied by procedures that do not alter the proton permeability of the thylakoid membranes. A linear relationship between the internal proton concentration and the rate of nonphosphorylating electron flow was previously verified. By determining the constant relating internal proton concentration to both ATP hydrolysis and electron flow, the proton/ATP ratio for the chloroplast ATPase complex was calculated to be 3.4 +/- 0.3. The presence of Pi, which allows ATP-Pi exchange to occur, lowers the internal proton concentration, but does not alter the relationship between the net rate of ATP hydrolysis and internal proton concentration. ATP-Pi exchange shows a dependence on the proton activity gradient very similar to that of ATP synthesis in the light. These results suggest that ATP-Pi exchange resembles photophosphorylation. In agreement with this idea, it is nucleoside diphosphate from the medium that is phosphorylated during exchange. Moreover, the energy-linked incorporation of Pi and ADP into ATP during exchange occurs at a similar rate. Thus, ATP synthesis from medium ADP and Pi takes place at the expense of the pH gradient generated by ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed1980

The onset of photophosphorylation correlates with the rise in transmembrane electrochemical proton gradients.

The onset of photophosphorylation was determined by exposing chloroplast thylakoids to either single or multiple light flashes of varying duration. In aggreement with the results of Ort et al. (Ort, D.R., Dilley, R.A. and Good, N.E. (1976) Biochim. Biophys. Acta 449, 108--124), the permeant buffer imidazole in the presence of valinomycin and K+ did not greatly delay the onset of phosphorylation driven by multiple activation. In single flashes, however, the lag in the development of phosphorylation was much longer and imidazole caused a further delay. A significant delta pH was generated by the multiple flash regime. The onset of photophosphorylation is, therefore, consistent with the rise in transmembrane delta pH.

Adenosine Triphosphate↗
PubMed1979

Reversible uncoupling of photophosphorylation by a new bifunctional maleimide.

A new bifunctional maleimide that contains a disulfide bond has been synthesized. This maleimide, dithiobis-N-ethylmaleimide (DTEM), like another bifunctional maleimide, o-phenylenebismaleimide, is about 500-fold more effective as an inhibitor of photophosphorylation than N-ethylmaleimide. Thylakoids must be illuminated in the presence of DTEM before the assay of phosphorylation for the inhibition to occur. Phosphoryalation in thylakoids treated with DTEM in the light is uncoupled and proton permeability of the treated thylakoids is enhanced. This uncoupling of photophosphorylation in thylakoids treated with DTEM can be reversed by thiol compounds. The addition of 50 mM dithiothreitol restores H+ uptake in thylakoids treated with DTEM in the light to control levels and partially reverses the inhibition of phosphorylation. Evidence is provided to show that DTEM cross-links groups within the gamma subunit of the coupling factor 1, and that the cross-link is broken by high concentrations of thiols. These results suggest that cross-linking is the cause for the increased proton permeability in thylakoids treated with bifunctional maleimides in the light.

Chloroplasts↗
PubMed1979

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark.

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg2+. The reaction is inhibited by (I) an antibody to ferredoxin-NADP+ reductases (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q. It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.

Chloroplasts↗
PubMed1978

How cells make ATP.

Explore the source record for details and available documents.

Adenosine Triphosphate↗
PubMed1976

Light-induced exchange of nucleotides into coupling factor 1 in spinach chloroplast thylakoids.

The method of centrifugation of chloroplast thylakoids through silicone fluid, previously used to estimate the uptake of solutes by thylakoids, is shown to be an excellent method for measuring binding of nucleotides to thylakoids. This binding, which is probably an exchange (Harris, D. A. and Slater, E. C. (1975) Biochim. Biophys. Acta 387, 335-348), is enhanced by light and is sensitive to uncoupling. Half-maximal binding of adenosine 5'-triphosphate (ATP) or adenosine 5'-diphosphate (ADP) at 10 mjM was reached within less than 0.1 s. With illumination times sufficient to elicit maximal binding, saturation of the site(s) is approached at 20 muM nucleotide and dissociation constants of 5 muM and 7 muM were calculated for ADP and ATP, respectively. At saturation, the binding corresponds to 1 mol/mol of coupling factor 1 or less. Although the light-dependent binding of ADP does not require Mg2+, that of ATP is markedly enhanced by Mg2+. A 10-fold molar excess of guanosine di- or triphosphate or adenyl-5'-yl imidodiphosphate had little effect on the binding. Adenosine 5'-phosphosulfate, a competitive inhibitor of phosphorylation with respect to ADP, decreases the binding. Thylakoids, previously illuminated in the absence of added nucleotides, retain the capacity to bind ADP or ATP in the dark long after the H+ electrochemical gradient has decayed. The conformation of coupling factor 1 in darkened thylakoids following illumination in the absence of added nucleotides may thus differ from that in thylakoids either illuminated in the presence of nucleotides or kept in the dark. Approximately 20% of the ADP bound to coupling factor 1 in thylakoids is converted to ATP by a 2-s illumination. Bound inorganic phosphate, derived either from ATP or from inorganic phosphate itself, serves as the phosphoryl donor. Bound ADP may, therefore, be of catalytic significance in the mechanism of phosphorylation.

Adenosine Diphosphate↗
PubMed1976

A simple, quantitative approach to the coupling of photophosphorylation to electron flow in terms of proton fluxes.

A simple relationship between observed phosphorylation efficiencies (P/e ratios) and internal proton concentration in spinach chloroplast thylakoids has been derived. P/e ratios, varked by either changing the light intensity or by adding the energy transfer inhibitor, 4'-deoxyphlorizin, were found to change with internal proton concentration in accordance with this relationship. A quantitative prediction of the effect of uncouplers on the P/e ratio can probably also be made. By extrapolation of plots of observed P/e ratios against internal proton concentration divided by the overall rate of electron flow, a maximum intrinsic P/e of about 0.66 is obtained. Assuming that two protons appear inside thylakoids per electron transferred, a P/e ratio of 0.66 suggests that three internal protons are consumed for each ATP formed. Internal protons may be considered to be substrates for the phosphorylation reaction. Hill plots of phosphorylation rate vs. internal proton concentration also indicate that three protons are consumed for each ATP synthesized. Thus, the H+ concentration gradient behaves quantitatively, as well as qualitatively, as if it is the connecting link between electron flow and phosphorylation in illuminated thylakoids.

Chloroplasts↗
PubMed1976

Acid-induced phosphorylation of adenosine 5'-diphosphate bound to coupling factor 1 in spinach chloroplast thylakoids.

Adenosine 5'-diphosphate, bound to coupling factor 1 (CF1) in spinach chloroplast thylakoids, is in part converted to adenosine 5'-triphosphate, upon injection of the thylakoids into strong acids in the dark. Bound phosphate serves as the phosphoryl donor for this uncoupler-insensitive conversion. Exposure of the thylakoids to heat or to urea prior to their injection into acid caused dissociation of ADP and prevents the apparent acid-induced synthesis of ATP. Conformational changes in CF1 may be elicited by acid denaturation which resemble those brought about by the proton electrochemical gradient across thylakoid membranes.

Adenosine Diphosphate↗
PubMed1976

Quantitative relationships between phosphorylation, electron flow, and internal hydrogen ion concentrations in spinach chloroplasts.

1. Further evidence that the uptake of [14C]hexylamine, determined by centrifugal filtration of spinach chloroplast thylakoids through silicone fluid layers, gives precise estimations of light-induced H+ concentration gradients (deltapH) is presented. DeltapH was independent of the amount of thylakoids used or of the concentration of hexylamine. Moreover, hexylamine uptake was sensitive to the osmolarity of the suspending medium. 2. Internal H+ concentration ([H+]in) is proportional to the rate of electron flow when light intensity was used to vary these parameters. Proportionality was still observed in the presence of 0.1 and 1.0 muM gramicidin D. When, however, [H+]in and electron flow were varied by increasing the concentration of gramicidin D, at constant light intensity the rate of electron flow was approximately proportional to 1/[H]in. 3. The phosphorylation efficiency (P/e2 ratio) falls with decreasing light intensity or increasing concentrations of the phosphorylation inhibitor, 4'-deoxyphlorizin. The proportionality between the rate of electron flow and [H+]in allows the calculation of the rate of nonphosphorylating (basal) electron flow if [H+]in under phosphorylating conditions is known. The contribution of basal electron flow, a consequence of passive efflux of H+ from the thylakoids, to the overall rate of electron flow increases as the rate of phosphorylation decreases. P/e2 ratios calculated using rates of electron flow from which the basal component has been subtracted are constant. A calculated P/e2 ratio of about 1.3 is obtained. 4. It is shown that the reciprocal of the phosphorylation efficiency should be proportional to 1/[H+]in2 when these parameters are varied using light intensity. This relationship was verified and provided an estimate of the P/e2 at infinite [H+]in. This value was 1.3. These results provide further evidence that a H+ electrochemical gradient serves to couple photophosphorylation to electron flow and that the rate of phosphorylation is proportional to [H+]in3. That is, three H+ are translocated out of thylakoids for each adenosine triphosphate formed.

Chlorophyll↗
PubMed1976

Role of Galactolipids in Spinach Chloroplast Lamellar Membranes: II. Effects of Galactolipid Depletion on Phosphorylation and Electron Flow.

A galactolipid lipase from primary bean (Phaseolus vulgaris) leaves has been used to partially deplete spinach chloroplast inner membranes of their galactolipids. Chloroplasts treated with the lipase in the absence of bovine serum albumin lost 91% of their monogalactosyl diglyceride, 83% of their digalactosyl diglyceride, all of their phosphatidyl choline, but none of their sulfolipid. Electron microscopy of this sections revealed that the treated chloroplasts were greatly enlarged and lacked membrane stacking. Linolenic acid had similar effects on the structure of the chloroplasts. Chlorophyll, carotenoids, and coupling factor 1 remained bound to the treated membranes.To minimize the inhibition of phosphorylation and electron flow by fatty acids released by the lipase, bovine serum albumin (15-24 mg/ml) was added to the lipase incubation mixtures. Bovine serum albumin inhibited the extent, but not the initial rate, of fatty acid release by the lipase. Electron microscopy of chloroplasts treated with the lipase in the presence of bovine serum albumin showed that membrane stacking was partially maintained. Chloroplasts treated with lipase under these conditions retained about 30% of their monogalactosyl diglyceride, 50% of their digalactosyl diglyceride and phosphatidyl choline. The sulfolipid and phosphatidyl glycerol contents were unchanged. Electron flow through photosystems I and II with artificial electron donors and acceptors was not affected by lipase treatment in the presence of bovine serum albumin. In contrast, oxygen evolution and phosphorylation were partially inhibited. These reactions are also very sensitive to fatty acids and it is possible that the inhibition is the result of interaction of fatty acids with the membrane prior to their binding to bovine serum albumin.In view of the irreversible inactivation of electron flow and phosphorylation by fatty acids, it is difficult to assess the role of galactolipids in these processes when a specfic lipase is used to deplete the membrane.

Journal Article↗
PubMed1975

Influence of adenine nucleotides on the inhibition of photophosphorylation in spinach chloroplasts by N-ethylmaleimide.

The incubation of spinach chloroplasts with 1 mM N-ethylmaleimide in light for 60 to 90 s results in a partial, irreversible inhibition of photophosphorylation. The inhibition was not overcome at infinite light intensity or at infinite concentrations of the phosphorylation substrates. Although the inhibition diminished with decreasing concentrations of adenosine diphosphate in the assay of phosphorylation, the inhibition of guanosine diphosphate phosphorylation was independent of the concentration of this nucleotide. Although adenosine di- or triphosphate (10 to 30 muM) alone partially prevented the development of the N-ethylmaleimide inhibition of phosphorylation, these nucleotides were more effective when either 1 mM inorganic phosphate or arsenate was also present. The light-dependent incorporation of N-ethylmaleimide into chloroplast-bound coupling factor 1 was affected by adenosine triphosphate and inorganic phosphate in a manner similar to the onset of N-ethylmaleimide inhibition. Since guanosine diphosphate did not protect phosphorylation from N-ethylmaleimide inhibition but is phosphorylated at rapid rates, it is apparent that coupling factor 1 in chloroplasts has multiple nucleotide recognition sites.

Adenine Nucleotides↗