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R E McCarty

Publications and source records attributed to R E McCarty.

18 recordsLinked to original sources
PubMed1992

Modifications of the gamma subunit of chloroplast coupling factor 1 alter interactions with the inhibitory epsilon subunit.

The treatment of chloroplast coupling factor 1 (CF1) with dithiothreitol or with trypsin modifies the gamma subunit. Reduction of the gamma subunit disulfide bond in CF1 in solution with dithiothreitol enhances the dissociation of epsilon (Duhe, R. J., and Selman, B. R. (1990) Biochim. Biophys. Acta 1017, 70-78). The Ca(2+)-ATPase activity of either oxidized or reduced CF1 increases as the enzyme is diluted. Added epsilon subunit inhibits the Ca(2+)-ATPase activity of both forms of the diluted CF1, suggesting that epsilon dissociation is the cause of activation by dilution. Half-maximal activation occurred at much higher concentrations of the reduced CF1, indicating that reduction decreases the affinity for epsilon about 20-fold. Immunoblotting techniques show that there is only one epsilon subunit/CF1 in intact chloroplasts, in thylakoid membranes, and in solution. No epsilon is released from CF1 in thylakoids under conditions of ATP synthesis. The gamma subunit of CF1 in illuminated thylakoids is specifically cleaved by trypsin. CF1 purified from thylakoids treated with trypsin in the light is deficient in epsilon subunit, and has a high rate of ATP hydrolysis. Added epsilon neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.

Adenosine Triphosphate↗
PubMed1991

Fluorescence resonance energy transfer mapping of the fourth of six nucleotide-binding sites of chloroplast coupling factor 1.

Equilibrium dialysis measurements of adenine nucleotide binding to chloroplast coupling factor 1 suggest that the enzyme has six binding sites for ADP, adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), and 2'(3')-O-2,4,6-trinitrophenyl-ATP (TNP-ATP). High affinity binding at all six sites requires the divalent cation, Mg2+. Three of the nucleotide-binding sites, sites 1, 2, and 3, have been mapped by fluorescence resonance energy transfer distance measurements (see McCarty, R. E., and Hammes, G. G. (1987) Trends Biochem. Sci. 12, 234-237). Using the same technique, we mapped the location of nucleotide-binding site 4, a tight, exchangeable site (Shapiro, A. B., Huber, A. H., and McCarty, R. E. (1991) J. Biol. Chem. 266, 4194-4200). Two arrangements of the energy transfer map of coupling factor 1 were found which are compatible with the available data. The two arrangements make different predictions about which sites interact in cooperative catalysis.

Adenosine Diphosphate↗
PubMed1991

Four tight nucleotide binding sites of chloroplast coupling factor 1.

We have examined the properties of the four tight nucleotide binding sites of reductively activated chloroplast coupling factor 1. Tight sites are here defined as those which retain bound nucleotides after passage of the chloroplast coupling factor 1 through Sephadex gel filtration centrifuge columns. Two of the sites, here called sites 4 and 5, have not been characterized in detail before. Site 4 has properties similar to those of site 1. It binds to ADP, ATP, and adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) tightly in the presence or absence of Mg2+. Bound ADP exchanges rapidly with medium ADP, but rapid exchange with ATP or AMP-PNP requires Mg2+. Site 4 may slowly hydrolyze bound ATP in the absence of medium nucleotides. Site 5 has properties similar to those of site 2. Tight binding of ATP and AMP-PNP requires Mg2+, but Mg29+)-ADP is not tightly bound. Site 5 does not hydrolyze bound ATP in the absence of medium nucleotides. Complete filling of all four tight nucleotide binding sites requires about one millimolar nucleotide, suggesting that low affinity binding sites are converted to tight binding via a nucleotide binding-induced conformational change.

Adenine Nucleotides↗
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↗
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↗