ATPase Activity Of CF1 Research Articles

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591–598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca 2+- and Mg 2+-ATPase activities of CF1 deficient in its inhibitory ε subunit (CF1-ε) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca 2+-ATPase activity by CTAB is irreversible. The Ca 2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg 2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-ε or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-ε from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the α/β heterohexamer.

Low Concentrations of Tetracycline Enhance the Photophosphorylation and P/O Ratio of Chloroplasts

This study investigated the effects of tetracycline on photophosphorylation, electron transport and P/O ratio of spinach chloroplasts. When chloroplast preparations were treated with low concentrations of tetracycline, non-cyclic and cyclic photophosphorylation activities increased, electron transport rates and P/O ratios improved, chloroplast ms-DLE also improved, and the Mg(2+)-ATPase activity of CF1 increased in comparison to the control. These results indicate that spinach chloroplasts are sensitive to tetracycline. Next, we used the fluorescence emission spectra of CF1 to examine the possible binding sites for tetracycline. The fluorescence emission spectra of CF1 treated with glutaraldehyde, NEM and TNBS, which interact with CF1 across its whole structure, at the γ subunit and at the β subunit, respectively, were compared with that of control CF1. The peak sites of the various fluorescence emission spectra were the same, but the peak values for CF1 treated with glutaraldehyde, NEM and TNBS were lower than that of control CF1. The peak value of CF1 treated with 50 µM tetracycline was very similar to that of CF1 treated with NEM. The above results indicate that the acting site of tetracycline may be at or near the γ subunit of CF1, and allows the creation of a model in which tetracycline binding strengthens the subunit interactions of ATP synthase, enlarges the proton motive force across the thylakoid membrane, and allows the excess proton motive force to increase ATP formation and improve the P/O ratio.

Interaction of oxyanions with thioredoxin-activated chloroplast coupling factor 1

Interaction between F 1-ATPase activity stimulating oxyanions and noncatalytic sites of coupling factor CF 1 was studied. Carbonate, borate and sulfite anions were shown to inhibit tight binding of [ 14C]ATP and [ 14C]ADP to CF 1 noncatalytic sites. The demonstrated change of their inhibitory efficiency in carbonate–borate–sulfite order coincides with the previously found change in efficiency of these anions as stimulators of CF 1-ATPase activity [Biochemistry (Mosc.) 43 (1978) 1206–1211]. Inhibition of tight nucleotide binding to noncatalytic sites was accompanied by stimulation of nucleotide binding to catalytic sites. This suggests that stimulation of CF 1-ATPase activity is caused by interaction between oxyanions and noncatalytic sites. A most efficient stimulator of CF 1-ATPase activity, sulfite oxyanion, appeared to be a competitive inhibitor with respect to ATP and a partial noncompetitive inhibitor with respect to ADP. The inhibition weakened with increasing time of CF 1 incubation with sulfite and nucleotides. Sulfite is believed to inhibit fast reversible interaction between nucleotides and noncatalytic sites and to produce no effect on subsequent tight binding of nucleotides. A possible mechanism of the oxyanion-stimulating effect is discussed.

Interaction of sulfite with the noncatalytic and catalytic sites of chloroplast coupling factor cf1.

The interaction between sulfite, an efficient Mg2+-dependent F1-ATPase activator, and chloroplast CF1-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF1. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF1 catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg2+-dependent ATPase activity of CF1 is caused by sulfite binding to its catalytic sites.

Structure function relationship of vanadate bound to a single site in chloroplast CF1-ATPase as determined by X-ray absorption

The structure of vanadate, a phosphate analogue which was suggested to function in the presence of tightly bound ADP and divalent cations as a transition state inhibitor of CF1-ATPase, was investigated by X-ray absorption spectroscopy. Analysis of the vanadium K-edge was used for determination of the structure of vanadate bound to a single site in CF1-ATPase containing a single tightly bound ADP. There was a decrease in the intensity of the 1s-3d pre-edge transition and a change in the shape of two other shoulders at the edge region upon binding of vanadate to CF1 in the presence of Mg2+ ions. The changes are due to alteration in the structure of vanadium from tetrahedral to a five-coordinated trigonal bipyramidal geometry. Comparison of the pre-edge peak intensity of ADP-vanadate complex, and model compound resolved by crystallography support the proposed structure of CF1-bound vanadate. 51V NMR measurements were used to verify the pentacoordinated structure of ADP-vanadate complex used as a model in the X-ray absorption studies. The inhibition of a single and multiple site activity by vanadate and by MgADP was measured. Vanadate inhibition of CF1-ATPase activity decreased more than 90 fold in the presence of MgADP. A differential specificity of the inhibition in single and multiple mode of activity was observed. It is suggested that ADP-vanadate binds to the active sites of the enzyme as a pentacoordinated vanadium having approximate trigonal bipyramidal geometry. This structure is analogous to the proposed transition state of the phosphate during the synthesis and the hydrolysis of ATP by CF1.

Functional Consequences of Deletions of the N Terminus of the [epsilon] Subunit of the Chloroplast ATP Synthase.

The [epsilon] subunit of the chloroplast ATP synthase functions in part to prevent wasteful ATP hydrolysis by the enzyme. In addition, [epsilon] together with the remainder of the catalytic portion of the synthase (CF1) is required to block the nonproductive leak of protons through the membrane-embedded component of the synthase (CFO). Mutant [epsilon] subunits of the spinach (Spinacia oleracea) chloroplast ATP synthase that lack 5, 11, or 20 amino acids from their N termini ([epsilon]-[delta]5N, [epsilon]-[delta]11N, and [epsilon]-[delta]20N, respectively), were overexpressed as inclusion bodies. Using a procedure that resulted in the folding of full-length, recombinant [epsilon] in a biologically active form, none of these truncated forms resulted in [epsilon] that inhibited the ATPase activity of CF1 deficient in [epsilon], CF1(-[epsilon]). Yet, the [epsilon]-[delta]5N and [epsilon]-[delta]11N peptides significantly inhibited the ATPase activity of CF1(-[epsilon]) bound to CFO in NaBr-treated thylakoids. Although full-length [epsilon] rapidly inhibited the ATPase activity of CF1(-[epsilon]) in solution or bound to CFO, an extended period was required for the truncated forms to inhibit membrane-bound CF1(-[epsilon]). Despite the fact that [epsilon]-[delta]5N significantly inhibited the ATPase activity of CF1(-[epsilon]) bound to CFO, it did not block the proton conductance through CFO in NaBr-treated thylakoids reconstituted with CF1(-[epsilon]). Based on selective proteolysis and the binding of 8-anilino-1-naphthalene sulfonic acid, each of the truncated peptides gained significant secondary structure after folding. These results strongly suggest (a) that the N terminus of [epsilon] is important in its binding to CF1, (b) that CF0 stabilizes [epsilon] binding to the entire ATP synthase, and (c) that the N terminus may play some role in the regulation of proton flux through CFO.

Differences between Two Tight ADP Binding Sites of the Chloroplast Coupling Factor 1 and Their Effects on ATPase Activity

Purified chloroplast ATP synthase (CF1) contains 1.2-2 mol of tightly bound ADP/mol of enzyme that resists removal by gel filtration or dialysis. CF1 was depleted of its endogenous nucleotide by treatment with alkaline phosphatase. Tightly bound nucleotide was demonstrated not to have an essential structural role. CF1 depleted of endogenous nucleotide retains its ability to catalyze Ca2+- and Mg2+-dependent ATPase activity and is not more sensitive to cold inactivation than untreated CF1. 2'(3')-O-Trinitrophenyladenosine 5'-diphosphate (TNP-ADP) binds tightly to two sites on nucleotide-depleted CF1, binding to either site at a faster rate than that of exchange of bound nucleotide for medium nucleotide. The nucleotide-depleted enzyme binds about one additional mol of TNP-ADP/mol of CF1, indicating that there is a tight TNP-ADP binding site that does not exchange readily with medium nucleotide. It is MgADP in this nonexchanging site, not the easily exchanging ADP binding site, that is responsible for the MgADP-induced inhibition of the ATPase activity. The rate of exchange of tightly bound ADP from CF1 matches the rate at which the Mg2+ATPase activity of CF1 is activated but is not itself responsible for the activation.

A role for the disulfide bond spacer region of theChlamydomonas reinhardtii coupling factor 1 γ-subunit in redox regulation of ATP synthase

The gamma-subunit of chloroplast coupling factor 1 contains a disulfide bond which is involved in the redox regulation of the enzyme. In all the sequence plant gamma-subunits this disulfide bond is separated by a five amino acid spacer region. To investigate the regulatory significance of this region genetic transformation experiments were performed with Chlamydomonas reinhardtii. C. reinhardtii strain atpC1 (nitl-305, cw 15, mt-), which does not accumulate the CF1 gamma-subunit polypeptide, was independently transformed with two constructs, each bearing mutations within the disulfide bond spacer region between Cys198 and Cys204 of the gamma-subunit. Successful complementation was confirmed by phenotypic selection, Northern blot analysis, and reverse transcription polymerase chain reaction. Whereas wild-type thylakoid membrane particles catalyze in vitro, PMS-dependent photophosphorylation that is stimulated 2-fold by the addition of DTT, similar particles from each of the mutant strains exhibit rates of ATP synthesis that are independent of DTT. Consistent with these results, wild-type CF1 ATPase activity is stimulated by DTT which is in contrast to the ATPase activities of both the mutant strains which are independent of DTT addition. These results suggest a role of the gamma-subunit disulfide bond spacer region in the redox regulation of chloroplast ATP synthase.

Alkylation of cysteine 89 of the gamma subunit of chloroplast coupling factor 1 with N-ethylmaleimide alters nucleotide interactions.

Alkylation of Cys-89 of the gamma subunit of the coupling factor portion (CF1) of the chloroplast ATP synthase by N-ethylmaleimide was previously shown to inhibit ATP synthesis and hydrolysis. The gamma subunit interacts with the inhibitory epsilon subunit. Alkylation of gamma Cys-89 could cause changes that result in the strengthening of the interactions between the two subunits which would be inhibitory. We show that the inhibition of the ATPase activity of CF1 in solution persists after removal of the epsilon subunit. Additionally, epsilon rebinds to control and alkylated CF1 at very similar CF1 to epsilon concentration ratios. Although the bound nucleotide contents of control and alkylated CF1 were similar, the rate of exchange of nucleotide bound to one site with medium nucleotide was accelerated by more than two orders of magnitude by alkylation. We show that ATP-induced release of bound 2'(3')-O-trinitrophenyl-adenosine 5'-diphosphate can be monitored conveniently by stopped-flow fluorescence. The effects of N-ethylmaleimide modification are likely to be felt over long distances. As determined by fluorescence resonance energy transfer, no nucleotide binding site so far mapped is closer than 5 nm to Cys-89. The bulky maleimide probably causes structural changes that perturb subunit and catalytic site interactions.

Catalytic properties of ? subunit isolated from chloroplast coupling factor 1

The chloroplast coupling factor (CF1) was dissociated into subunits by the freezing-thawing procedure in the presence of 0.5 M NaBr and the β subunit was purified by ion-exchange chromatography on a DEAE-cellulose column. The β subunit did not catalyze ATP hydrolysis either in the presence or in the absence of reagents known to activate Mg(2+)-dependent ATPase activity of CF1. However, it manifested appreciable adenylate kinase-like and ATP-ADP γ-phosphate exchange activities. The adenylate kinase-like activity only slightly depended on Mg(2+) ions. Ethanol, and especially diadenosine pentaphosphate, inhibited the reaction effectively. In contrast, the ATP-ADP exchange activity was Mg(2+)-dependent. Ethanol and diadenosine pentaphosphate were poor inhibitors. Sulfite, the CF1-ATPase activator, and quercetin, its inhibitor, had a minor effect on catalytic activity of the β subunit.

The dithiothreitol-stimulated dissociation of the chloroplast coupling factor 1 ϵ-subunit is reversible

The chloroplast coupling factor 1 complex (CF1) contains an ϵ-subunit which inhibits the CF1 ATPase activity. Chloroform treatment of Chlamydomonas reinhardtii thylakoid membranes solubilizes only forms of the enzyme which apparently lack the δ-subunit. Four interrelated observations are described in this paper. (1) The dithiothreitol- (DTT) induced ATPase activation of CF1(−δ) and the DTT-induced formation of a physically resolvable CF1(−δ,ϵ) from the CF1(−δ) precursor are compared. The similar time-courses of these two phenomena suggest that the dissociation of the ϵ-subunit is an obligatory process in the DTT-induced ATPase activation of soluble CF1. (2) The reversible dissociation of the ϵ-subunit of the CF1 is demonstrated by the exchange of subunits between distinguishable oligomers. 35S-labelled chloroplast coupling factor 1 lacking the δ and ϵ subunits [CF1(−δ,ϵ)] was added to a solution of non-radioactive coupling factor 1 lacking only the δ subunit [CF1(−δ)]. After separation of the two enzyme forms, via high resolution anion-exchange chromatography, radioactivity was detected in the chromatographic fractions containing CF1(−δ). (3) ϵ-deficient CF1 can be resolved from DTT pretreated ϵ-containing CF1 for several days after the removal of DTT. On the other hand, brief incubation of the DTT pretreated ϵ-containing CF1 with low concentrations of o-iodosobenzoate results in chromatographs containing only the peak of ϵ-containing CF1. A simple explanation for this phenomenon is that reduction of CF1 with DTT increases the apparent dissociation constant for the ϵ-subunit to an estimated 3.5 · 10−8 M (± 1.0 · 10−8 M) from a value of </ 5 · 10−11 M for the oxidized enzyme. (4) ATPase activity data show that oxidation of the ϵ-deficient enzyme does not completely inhibit its manifest activity, but oxidation of DTT pre-treated CF1 which contains the ϵ-subunit completely inhibits manifest activity. A simple model is proposed for the influence of the oxidation state of the soluble enzyme on the distribution of ATPase-inactive and ATPase-active subunit configurations.

ATP Hydrolysis Catalyzed by a β Subunit Preparation Purified from the Chloroplast Energy Transducing Complex CF1.CF0

Washing thylakoid membranes with 1 M LiCl causes the release of the beta subunit from the chloroplast energy transducing complex (CF1.CF0) in spinach chloroplasts. This protein purifies by size exclusion chromatography as a 180-kDa aggregate and, thus, is probably composed of a trimer of beta polypeptides. The purified aggregate binds ADP to a high and a low affinity site with dissociation constants of 15 and 202 microM, respectively. Mg2+ is required for ADP to bind to both sites. Manganese binds to the protein in a cooperative manner to at least two sites with high affinity. The beta subunit preparation catalyzes Mg2+-dependent ATP hydrolysis at rates which are comparable to other subunit-deficient CF1 preparations and is increased by treatments known to activate the Mg2+-ATPase activity of CF1. However, Ca2+ is not an effective cofactor for this reaction and treatments which activate the Ca2+-ATPase of CF1 are either ineffective or inhibitory.

Thiol modulation of the thylakoid ATPase. Lack of oxidation of the enzyme in the presence of [formula omitted] in vivo and a possible explanation of the physiological requirement for thiol regulation of the enzyme

(1) Illumination of Dunaliella induced an increase in the activity of CF 1-ATPase, and of fructose-1,6-bisphosphatase (FBPase), that could be assayed under standard conditions in subsequently lysed cells. The light-induced, but not the light-independent, activities could be prevented by inclusion of methyl viologen in the preillumination medium. This effect was concluded to be due to the prevention of reduction of ferredoxin by methyl viologen. (2) Markedly higher concentrations of added methyl viologen were required to prevent induction of ATPase than FBPase. Addition of 1 mM methyl viologen to the preillumination stage prior to illumination totally prevented the appearance of light-induced FBPase, whereas a concentration of 20 mM was required to prevent light-induction of ATPase completely. (3) Although methyl viologen added to intact algae in the light subsequent to achievement of steady-state light activation of ATPase and FBPase led to the inactivation of FBPase, light-induced ATPase was completely uninhibited. This effect indicates that whereas FBPase is subject to reversible modification by thiols in the light, thiol reduction of ATPase in vivo is irreversible in the light, and oxidation only occurs upon dissipation of Δ μ H + produced by darkening. (4) Stabilisation of the thiol-reduced form of CF 1 by Δ μ H + (Shahak, Y. (1985) J. Biol. Chem. 260, 1459–1464) accounts for the lower reducing pressure necessary to activate thiol-dependent ATPase, compared to FBPase. Implications for the regulation of CO 2 fixation are discussed. (5) Complete activation of CF 1-ATPase activity was induced by incubation of intact algae with dithiothreitol in the dark. It is suggested that thiol-regulation of the enzyme is necessary in vivo in order to prevent activation of an ATPase activity which would otherwise inevitably result from autocatalytic generation of Δ μ H + in the dark.

A study of factors which regulate the membrane appression of lettuce thylakoids in relation to irradiance

Factors that may influence the extent of thylakoid membrane appression have been examined using lettuce (Lactuca sativa cv. Celtuce) grown under different irradiances. Electron microscopy and salt-induced chlorophyll fluorescence suggest that the percentage of membrane appression is increased in plants grown in low light (20 Wm(-2)) compared with those grown in high light (150 Wm(-2)). In high light plants surface charge, as measured by 9-aminoacridine, was found to be twice that measured in low light plants. There was a similar difference in ATPase activity of CF1 and in light saturated photophosphorylation. The chlorophyll content of LHC-2 as a proportion of the total chlorophyll was greatest in thylakoids of low light plants. Measurement of non-cyclic photophosphorylation rates suggested that membrane appression has a stimulatory role in the photophosphorylation process. The importance of these inter-related factors for the mechanism of thylakoid appression is discussed.

Dependence of Flavonol Functions on Their Chemical Structure in Chloroplast Energy Reactions

The effect of a number of flavonols (quercetin, morin, quercetin-glucoside, quercetin-glucosylp-coumarate, kaempferol-glucoside, and kaempferol-glucosyl-p-coumarate) on electron and proton transfer reactions as well as on ATP synthesis and hydrolysis in pea chloroplasts has been studied. It was shown that NADP+ photoreduction and coupled photophosphorylation by chloroplasts were suppressed by flavonols within the concentration range of 10-5-10-4 M. The inhibitory effect was weakened as the structure of the substances became more complicated. The inhibition of ATPase activity of CF1 was decreased after glucosylation and acylation of the aglycones. Quercetin increased the values for proton uptake. On the contrary, QuGC and morin decreased H+ uptake and increased the proton gradient dissipation constant. Dissociation constants of flavonol OH-groups were calculated and experimentally determined. The dissociation constants of ionogenic groups were pK2, = 5.1; pK3, = 8.6; pK4, = 9.0-9.4; pK5 > 10; pK7 = 7.4-7.6. Flavonols form unstable complexes with metal ions in the presence of Mg2+ and Ca2+. At pK > 10 OH-groups take part in the complex formation. Regulatory aspects of flavonol function mechanism in chloroplast energy conserving reactions are discussed.

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.

Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF 1): The effect on the CF 1-ATPase activity

Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF 1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19–24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[β- 32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF 1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the β subunit of the CF 1 and since there are probably three β subunits per CF 1, these results indicate strong cooperativity among β subunits and between the site of tightly bound nucleotides and the catalytic sites.

Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase.

The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.

Preparation of an ϵ-deficient chloroplast coupling factor 1 having a high ATPase activity

A modification of the chloroform extraction preparation of CF 1, by elevating the pH during the DEAE-Sephadex purification step to 7.8 yields a 5-subunit CF 1. Different 4-subunit CF 1 preparations deficient in either δ- or ϵ-subunits, as well as pure β-subunits are obtained by fractionation of intact CF 1 complex by anion-exchange high-performance liquid chromatography. Unlike the 5-subunit latent enzyme, ϵ-deficient CF 1 has high Ca-ATPase activity which can be inhibited by purified ϵ-subunits. After trypsin or octylglucoside activations all preparations have identical high ATPase activity. These results suggest that the subunit regulates CF 1-ATPase activity.

The interaction of N,N'-dicyclohexylcarbodiimide with chloroplast coupling factor 1.

1. Incubation of soluble spinach Coupling Factor 1 (CF1) with dicyclohexylcarbodiimide (DCCD) results in the inactivation of the ATPase. The DCCD inactivation is time- and concentration-dependent. Complete inactivation of the CF1-ATPase activity requires the binding of 2 mol of DCCD/mol of CF1. The binding sites of DCCD are located on the beta subunit of CF1. 2. DCCD modification of soluble CF1 eliminates one adenine nucleotide binding site which is exposed by dithiothreitol activation or by incubation with tentoxin. The inactivation of both the ATPase activity and the adenine nucleotide binding site are pH-dependent. The inactivation of both the ATPase activity and the adenine nucleotide binding site are pH-dependent. Half-maximal inhibition occurs at about pH 7.5. 3. The DCCD-modified CF1, reconstituted with EDTA-treated chloroplasts, is fully active is restoring proton uptake but not in restoring ATP synthesis or light-dependent adenine nucleotide exchange.