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R E McCarty

Publications and source records attributed to R E McCarty.

At least 19 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↗
PubMed1990

Chromatographic purification of the chloroplast ATP synthase (CF0-CF1) and the role of CF0 subunit IV in proton conduction.

Chromatographic procedures were developed to purify chloroplast ATP synthase (CF0-CF1) in large amounts and to resolve subunits from this enzyme. The ATP synthase thus obtained has high ATP-Pi exchange and Mg2(+)-ATPase activities upon incorporation into asolectin liposomes. The purity of this preparation was about 95%. By modifications of this chromatographic procedure, we purified subunit IV-deficient CF0-CF1, subunit IV-deficient CF0, and subunit IV. Both ATP-Pi exchange and Mg2(+)-ATPase activities were impaired by depletion of subunit IV from CF0-CF1. Partial restoration of these activities was obtained by reconstituting subunit IV-deficient CF0-CF1 with subunit IV. The impairment of these activities was likely caused by a loss in proton conductivity of CF0 upon removal of subunit IV. The dicyclohexylcarbodiimide-sensitive Mg2(+)-ATPase of subunit IV-deficient CF0-CF1 was not as sensitive to the depletion of subunit IV as ATP-Pi exchange. Nearly 90% of subunit IV could be removed, but Mg2(+)-ATPase activity was inhibited by only 40-60%. Thus subunit IV of CF0-CF1 may not participate directly in proton transfer but may have a role in organizing and/or stabilizing CF0 structure.

Adenosine Triphosphate↗
PubMed1990

Subunit interactions within the chloroplast ATP synthase (CF0-CF1) as deduced by specific depletion of CF0 polypeptides.

The proton-linked ATP synthase (CF1-CF0) of chloroplasts consists of a catalytic component (CF1) and a membrane-embedded part (CF0) that interacts with CF1 and contains a proton channel. The subunits of CF0 which are involved in binding of CF1 were studied by examining the effect of selective depletion of subunits I, II, and IV of CF0 from the chloroplast ATP synthase on the association of the remaining CF0 subunits with CF1. Dissociated CF0 subunits were identified by sucrose density gradient centrifugation. Removal of subunit IV alone from CF0-CF1 did not cause dissociation of the other CF0 subunits from CF1. Upon removal of both subunits I and IV from CF0-CF1, subunit II also dissociated, but subunit III was still bound to CF1. Thus, at least two subunits of CF0, I and III, directly associate with CF1. Subunit II is unlikely to bind CF1 directly and may associate with subunit I. Although depletion of subunit IV does not cause dissociation of CF0 from CF1, its interaction with CF1 subunits is uncertain.

Chloroplasts↗
PubMed1990

Purification and reconstitution of active chloroplast F0.

Chloroplast F0 (CF0) was purified from the ATP synthase by Zwittergent 3-12 treatment and DEAE-Trisacryl anion exchange chromatography. Purified CF0 contains four subunits corresponding to subunits I, II, III, and IV. CF0 mediated proton translocation across the membrane after incorporation into asolectin liposomes. The CF0-mediated proton transport was inhibited by N,N'-dicyclohexylcarbodiimide and the binding of chloroplast coupling factor 1 (CF1). Rebinding of CF1 to CF0 liposomes resulted in reconstitution of N,N'-dicyclohexylcarbodiimide and uncoupler sensitive energy-transducing activities. Like CF0 in native thylakoid membranes, purified CF0 bound CF1 as well as CF1 deficient in either the delta or epsilon subunits.

Adenosine Triphosphate↗
PubMed1990

Substrate binding-induced alteration of nucleotide binding site properties of chloroplast coupling factor 1.

Two adenine nucleotide binding sites of chloroplast coupling factor 1 (CF1) were shown previously to switch their properties after exposure of the enzyme to Mg2(+)-ATP or Ca2(+)-ATP (Shapiro, A. B., and McCarty, R. E. (1988) J. Biol. Chem. 263, 14160-14165). The change in binding properties was monitored by fluorescence resonance energy transfer between Lucifer Yellow vinyl sulfone covalently bound to one alpha subunit and trinitrophenyl-ATP (TNP-ATP) tightly bound to nucleotide binding site 1. When the nucleotide binding properties of sites 1 and 3 switch during catalysis, site 3, which is nearer Lucifer Yellow than site 1, switches its nucleotide binding properties with site 1, allowing TNP-ATP to become tightly bound near Lucifer Yellow. The smaller separation allows energy transfer to occur, resulting in decreased Lucifer Yellow fluorescence. In this paper, we show that adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) bound to CF1 and caused nucleotide binding sites 1 and 3 to switch properties, but was not hydrolyzed. Using AMP-PNP, we also found that relaxation of the properties of the sites to the precatalysis state after removal of substrate occurred in the absence of hydrolysis of the last bound nucleotide. When Mg2+ was omitted during exposure of CF1 to ATP, there was very little hydrolysis or nucleotide site switching. When Mg2+ was added to a very low concentration which was more than stoichiometric with CF1, however, site switching occurred at its maximal level with virtually no increase in ATP hydrolysis. These results support a model in which binding of substrate Mg2(+)-ATP, not hydrolysis, causes the putative catalytic sites to switch properties, in agreement with the alternating site catalytic cooperativity hypothesis (Boyer, P. D. (1989) FASEB J. 3, 2164-2178). TNP-ATP, the fluorescence acceptor, did not cause nucleotide site switching when incubated with CF1 in the presence of EDTA to eliminate free Mg2+. Two possible additional nucleotide binding sites were detected, in addition to the three well characterized sites. At least one of these sites was close to the Lucifer Yellow site, judging by the amount of energy transfer caused by partial occupancy with TNP-ATP.

Adenine Nucleotides↗
PubMed1988

Alteration of the nucleotide-binding site asymmetry of chloroplast coupling factor 1 by catalysis.

Fluorescence resonance energy transfer was used to show that ATP hydrolysis induces a change in the properties of two nucleotide-binding sites in isolated chloroplast coupling factor 1 (CF1). The fluorescence donor was Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide- 3,6-disulfonate), covalently bound to a unique site on the alpha subunit between nucleotide-binding sites 2 and 3. The fluorescence acceptor was the ATP analog 2'(3')-trinitrophenyladenosine 5'-triphosphate (TNP-ATP), incorporated specifically into nucleotide-binding site 1. Energy transfer from Lucifer Yellow to TNP-ATP in site 1 was greater if catalysis occurred before TNP-ATP was incorporated than if no catalysis occurred, indicating that one of the nucleotide-binding sites near the Lucifer Yellow had changed its properties to those of site 1 as a result of catalysis. The amount of energy transfer increased with the degree of substrate excess during catalysis, as expected if catalysis were required for the new site 1 location. ADP, which binds to CF1, but is not a substrate for hydrolysis, caused little energy transfer. Titration of site 3 with TNP-ATP showed greater energy transfer from Lucifer Yellow when catalysis had not occurred, indicating that sites 1 and 3 switched properties as a result of catalysis. The amount of energy transfer declined exponentially with time between removal of substrate and addition of TNP-ATP to site 1, with a half-time of 1.5-2 h at room temperature. This result suggests that the change that results in switching of nucleotide-binding sites 1 and 3 relaxes in the absence of substrate. Our results show that the asymmetry of the nucleotide-binding sites of CF1 is not a permanent feature of the molecule.

Adenosine Triphosphate↗
PubMed1987

Energy-dependent changes in the conformation of the epsilon subunit of the chloroplast ATP synthase.

Rabbit antiserum raised against the isolated native epsilon subunit of the chloroplast coupling factor 1 activated the ATPase activity of coupling factor 1 in solution by removing the epsilon subunit. Incubation of thylakoid membranes with the antiserum in the dark had no effect on photophosphorylation or on the dithiothreitol-induced Mg2+-ATPase activity. Incubation with the antiserum during illumination, however, strongly inhibited both activities and caused the membranes to become leaky to protons. The results indicate that the formation of a proton gradient across the thylakoid membrane induces a change in conformation of the epsilon subunit of the ATP synthase such that it becomes susceptible to attack and removal by the antibodies. This change may be a part of the mechanism that results in energy-dependent activation of the ATP synthase.

Ca(2+) Mg(2+)-ATPase↗
PubMed1986

Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1.

A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1). The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed. The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores. The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R. rubrum Fo-F1 complex. The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin. In contrast, chromatophores reconstituted with the native R. rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin. These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes. They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form.

Adenosine Triphosphate↗
PubMed1985

Binding stoichiometry and structural mapping of the epsilon polypeptide of chloroplast coupling factor 1.

Fluorescent probes were attached to the single sulfhydryl residue on the isolated epsilon polypeptide of chloroplast coupling factor 1 (CF1), and the modified polypeptide was reconstituted with the epsilon-deficient enzyme. A binding stoichiometry of one epsilon polypeptide per CF1 was obtained. This stoichiometry corresponded to a maximum inhibition of the Ca2+-dependent ATPase activity of the enzyme induced by epsilon removal. Resonance energy transfer between the modified epsilon polypeptide and fluorescent probes attached to various other sites on the enzyme allowed distance measurements between these sites and the epsilon polypeptide. The epsilon-sulfhydryl is nearly equidistant from both the disulfide (23 A) and the dark-accessible sulfhydryl (26 A) of the gamma subunit. Measurement of the distance between epsilon and the light-accessible gamma-sulfhydryl was not possible due to an apparent exclusion of modified epsilon from epsilon-deficient enzyme after modification of the light-accessible site. The distances measured between epsilon and the nucleotide binding sites on the enzyme were 62, 66, and 49 A for sites 1, 2, and 3, respectively. These measurements place the epsilon subunit in close physical proximity to the sulfhydryl-containing domains of the gamma subunit and approximately 40 A from the membrane surface. Enzyme activity measurements also indicated a close association between the epsilon and gamma subunits: epsilon removal caused a marked increase in accessibility of the gamma-disulfide bond to thiol reagents and exposed a trypsin-sensitive site on the gamma subunit. Either disulfide bond reduction or trypsin cleavage of gamma significantly enhanced the Ca2+-ATPase activity of the epsilon-deficient enzyme. Thus, the epsilon and gamma polypeptides of coupling factor 1 are closely linked, both physically and functionally.

Binding Sites↗
PubMed1985

Partial proteolysis as a probe of the conformation of the gamma subunit in activated soluble and membrane-bound chloroplast coupling factor 1.

Treatments that enhance the latent ATPase activity of the chloroplast coupling factor (CF1) also induce hypersensitivity of the gamma subunit toward trypsin. A number of different gamma subunit cleavage products are formed (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). We have compared the gamma cleavage products of membrane-bound and isolated CF1, activated either by reduction of the gamma disulfide bond or by removal of the epsilon subunit. The gamma subunit of isolated CF1 lacking the epsilon subunit was cleaved to a 27,000-Da species. The same cleavage site became exposed following energy-dependent conformational changes in the membrane-bound enzyme. Activation by reduction of the gamma disulfide bond also exposed this site. However, the gamma subunit of reduced CF1 was cleaved rapidly at an additional site and trypsin treatment gave rise to a 25,000-Da gamma species. The small peptide generated by the second cleavage contains one of the cysteinyl residues of the reduced disulfide bridge of gamma. This peptide dissociates from the enzyme and can be isolated by gel filtration. The close proximity of the trypsin cleavage sites to the disulfide bond of gamma is discussed with respect to the effects of tryptic cleavage on the ATPase activity of CF1. The data indicate that structural changes in a limited region of the gamma subunit strongly influence the catalytic properties of both soluble and membrane-bound CF1.

2-Naphthylamine↗
PubMed1985

Selective modification of an alpha subunit of chloroplast coupling factor 1.

Lucifer yellow (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulf onate), a fluorescent probe that can react covalently with sulfhydryl or amino groups, has been used to modify chloroplast coupling factor 1 (CF1). Conditions are described under which Lucifer yellow selectively labels the alpha subunit of CF1 to the extent of about 1 mol of probe per mole of CF1. An especially reactive amino group is apparently labeled, and modification has little effect on the ATPase activity of the enzyme. Lucifer yellow is a useful probe for fluorescence energy transfer measurements. The distances between this probe and fluorescent and absorbing molecules attached to seven specific sites on the beta, gamma, and epsilon subunits were determined. These distances converge to a single location. In addition to providing further information about the structure of CF1, these results suggest that the alpha subunits of CF1 are not structurally equivalent.

Chromatography, High Pressure Liquid↗
PubMed1985

Detergent activation of the ATPase activity of chloroplast coupling factor 1.

The activation of the ATPase activity of coupling factor 1 (CF1) from chloroplasts by several detergents was studied. Further evidence that detergent micelles are important in the activation of Ca2+-ATPase was obtained. Maximal activation of CA2+-ATPase was achieved with short-chain alkyl-beta-D-glucopyranoside (alkylglucosides) detergents. Treatment of CF1 with hexylglucoside or heptylglucoside followed by hydroxylapatite chromatography caused nearly total removal of the epsilon subunit of the enzyme, whereas treatment with decylglucoside caused less ATPase activation and less loss of the epsilon subunit. The ATPase activity of detergent-activated CF1 was inhibited by purified epsilon subunit. Detergents that form small micelles appear to be most effective in removing the epsilon subunit and in activating the Ca2+-ATPase of CF1. When present during assay, the alkylglucosides also induce a Mg2+-ATPase activity in CF1. Octyl- and nonylglucoside are most effective in promoting this reaction. If, however, CF1 deficient in the epsilon subunit was used, even decylglucoside elicited rapid Mg2+-ATPase hydrolysis. It is concluded that removal of the epsilon subunit, although necessary for the expression of Mg2+-ATPase, is not sufficient. The detergents that cause maximal displacement of the epsilon subunit are less effective in inducing Mg2+-ATPase activity. The selective removal of subunits from CF1 by specific detergents points to potential problems with the use of these detergents in the solubilization of oligomeric membrane proteins.

Adenosine Triphosphatases↗
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↗