Tentoxin-induced energy-independent adenine nucleotide exchange and ATPase activity with chloroplast coupling factor 1.

1. The effect of energy transfer inhibitors on energy-dependent exchange of tightly bound adenine nucleotides with washed, broken spinach thylakoids has been studied. Energy transfer inhibitors that inhibit the ATPase activity of soluble chloroplast coupling factor 1 (CF1) (e.g. phloridzin and tentoxin) do not inhibit energy-dependent adenine nucleotide exchange. Energy transfer inhibitors that block proton flux through the hydrophobic protein proton channel (CF0) (e.g. dicyclohexylcarbodiimide and triphenyltin chloride) also block light-dependent adenine nucleotide exchange. 2. Tentoxin, at relatively high concentrations, stimulates an energy-independent exchange of adenosine diphosphate. 3. High concentrations of tentoxin elicit a Ca2+-dependent ATPase activity with soluble CF1, but has no effect on the Ca2+-dependent ATPase activity of membrane-bound CF1. 4. The trypsin-activated, Ca2+-dependent, membrane-bound ATPase is not affected by high concentrations of tentoxin, whereas the dithiothreitol-activated, Mg2+-dependent ATPase is markedly inhibited. 5. The reconstitution of chloroplasts, partially depleted in CF1, with soluble CF1 is correlated with the loss of tentoxin-induced, Ca2+-dependent ATPase activity associated with soluble CF1.     

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.     

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.     

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.     

Lack of correlation between effects of tentoxin on chloroplast coupling factor and chloroplast ultrastructure

The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF₁) ATPase activity was investigated through an examination of the effects of tentoxin on electrophoretically-separated CF₁ ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes"upon electrophoresis but none of the isozymes were tentoxin sensitive. CF₁ isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. & Gray and the CF₁-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF¹ for tentoxin-induced chlorosis.     

Partial resolution of the enzymes catalyzing photophosphorylation. XV. Approaches to the active site of coupling factor I.

1. Prolonged treatment of coupling factor I (CF1) from spinach chloroplasts with trypsin free of chymotrypsin yielded an active ATPase. The isolated preparation showed only two polypeptide chains (mol wt 55,000 to 60,000) on acrylamide gels run in the presence of sodium dodecyl sulfate. The three smaller subunits of CF1 were not detectable. The preparation no longer served as a coupling factor for photophosphorylation in either EDTA- or silicotungstate-treated chloroplasts. 2. An antiserum prepared against coupling factor I from chloroplasts inhibited the ATPase activity of the trypsin-treated CF1. In contrast, antisera prepared against the two individual (denatured) subunits did not inhibit the ATPase activity when tested either alone or together, although each interacted with the trypsin-treated protein, forming precipitin lines in Ouchterlony plates. 3. The trypsin-treated enzyme was still cold-labile, showing that the three smaller subunits are not required for this property. However, the enzyme was no longer sensitive to the natural inhibitor protein which is one of its subunits (subunit epislon), but was still sensitive to inhibition by the flavonoid quercetin. 4. Two equivalents of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole were sufficient to inhibit about 80% of the ATPase activity of the coupling factor, irrespective of whether it contained two of five subunits. The inhibition was completely reversed by dithiothreitol. 5. Triated 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was prepared. Treatment of the coupling factor with this tritium-labeled inhibitor followed by electrophoresis on acrylamide gels revealed that most of the radioactivity was incorporated into the beta subunit of the enzyme (molecular weight 56,000).     

Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. I. Electrophoretic characterization and reconstitution of photophosphorylation

The coupling factor ATPase (F1) from photosynthetic membranes of the cyanobacterium Spirulina platensis was purified to homogeneity by a combination of ion-exchange chromatography and sucrose density gradient centrifugation. The ATPase activity of purified Spirulina F1 is latent but can be elicited by trypsin treatment, resulting in specific activities (CaATPase) of 27-37 mumol Pi min-1 mg protein-1. On denaturing sodium dodecyl sulfate-polyacrylamide gradient gels, Spirulina F1 is resolved into five subunits with molecular weights of 53,400, 51,600, 36,000, 21,100, and 14,700, similar to the molecular weights of the subunits of spinach chloroplast coupling factor (CF1). As determined by native polyacrylamide gradient gel electrophoresis, the molecular weight of the Spirulina F1 holoenzyme was estimated to be 320,000, somewhat smaller than the estimated molecular weight of spinach CF1 (392,000). Spirulina F1 was shown to be an active coupling factor by its ability to reconstitute phenazine methosulfate-dependent cyclic photophosphorylation in membrane vesicles which had been depleted of coupling factor content by 2 M NaBr treatment. We estimate the Spirulina F1 content of membrane vesicles to be 1 F1 per 830 chlorophylls or 0.12 mol F1 mol P700(-1), based on the specific ATPase activities of the membrane vesicles and the purified Spirulina F1, the molecular weight of F1, and the P700 content of the vesicles.     

Quercetin interaction with the chloroplast ATPase complex

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF1 and the ATPase activity of isolated CF1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexylcarbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H+ uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H+ uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photophosphorylation is discussed, and interaction with both CF1 and F0 is suggested.     

Effects of organic solvents and tentoxin on enzyme-bound ATP synthesis in isolated chloroplast coupling factor 1

Isolated spinach CF1 (chloroplast coupling factor 1) forms enzyme-bound ATP without any supply of energy in the presence of high concentrations of Pi [Feldman and Sigman (1982) J Biol Chem 257: 1676-1683]. The final amount of CF1-bound ATP synthesized was increased greatly by 1,2-propanediol, and moderately by methanol, ethanol, and dimethyl sulfoxide, but decreased by glycerol and octyl glucoside. Methanol and ethanol greatly increased the initial rate of ATP synthesis, while 1,2-propanediol increased it only moderately. Low concentrations (10^-8 -10^-6 M) of tentoxin, which inhibit ATPase activity of isolated CF1, did not affect enzyme-bound ATP synthesis. However, high concentrations (>10^-5 M) of tentoxin, which stimulate ATPase activity of isolated CF1, enhanced the initial rate of CF1-bound ATP synthesis without significant effect on the final amount of ATP synthesized in the presence of medium ADP. The substrate of enzyme-bound ATP synthesized came largely from tightly bound ADP, not medium ADP, and tentoxin did not affect this substrate profile. Tentoxin did not affect the binding of medium ADP to high affinity sites on CF1.     

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach

The effects of nano-TiO₂ (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO₂, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg²⁺-ATPase and chloroplast coupling factor I (CF₁)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO₂ might be related to activation of photochemical reaction of chloroplasts of spinach.     

Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.     

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts.

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: (a) the disappearance of three out of four accessible thiol groups, two from gamma and one from a beta subunit as a consequence of CF1 modification by o-iodosobenzoate; (b) the total free sulphydryl groups of CF1 were reduced from 8 to 4 after modification of CF1 by o-iodosobenzoate. Two groups disappeared from beta and two from gamma subunits; (c) a second heating step of CF1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from beta and gamma subunits.     

The special reaction of photophosphorylation using epsilon ADP--a fluorescent analog of ADP

Photophosphorylation of epsilon ADP in a chloroplast synthetase system reconstituted with CF1 or with CF1 modified by covalently bound epsilon ADP has been studied. The reconstitution of EDTA-treated chloroplasts with CF1 restores the photophosphorylating activity to about 90%. When the CF1 modified by covalently bound epsilon ADP is used for reconstitution the photophosphorylating activity of EDTA-treated chloroplasts is restored to 37%. Based on the results of a photochemical study of the chloroplast ATP-synthetase system reconstituted with CF1 with covalently bound epsilon ADP it may be assumed that the substrate, adenine, participates in proton translocation to inorganic phosphate in the active center of the coupling enzyme during photophosphorylation.     

Role of the gamma subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol

In leaves and intact chloroplasts, oxidation and reduction have been shown previously to regulate the ATPase activity of thylakoids. Illumination of spinach chloroplast thylakoids in the presence of dithiothreitol, which activates the ability of thylakoids to catalyze sustained ATP hydrolysis in the dark, causes increased incorporation of N-ethylmaleimide into the gamma subunit of coupling factor 1 (CF1). A disulfide bond in the gamma subunit is reduced during activation. The residues involved in this disulfide bond are the same as those in the disulfide linkage reduced during dithiothreitol activation of soluble CF1. The disulfide and dithiol forms of the gamma subunit may be separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. N-Ethylmaleimide is preferentially incorporated in the dark into the reduced form of the gamma subunit of CF1 in thylakoids previously exposed to dithiothreitol. Only a subpopulation of the CF1 in thylakoids is susceptible to dithiothreitol reduction and subsequent reaction with N-ethylmaleimide in the dark. Alkylation of the thiol groups exposed by reduction of the disulfide bond protects ATPase activity from inhibition by oxidants. At a given value of the transmembrane pH differential, photophosphorylation rates in dithiothreitol-activated thylakoids can be as much as seven to eight times those of nonactivated controls. N-Ethylmaleimide treatment of activated thylakoids in the dark prevents the loss of the stimulation of ATP synthesis on storage of the thylakoids. Photophosphorylation by intact chloroplasts lysed in assay mixtures is also activated in comparison to that by washed thylakoids. At a low ADP concentration, the rate of photophosphorylation approaches saturation as delta pH increases. These results suggest that the gamma subunit of CF1 plays an important role in regulation of ATP synthesis and hydrolysis.     

Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin.

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF1) as well as several hydrophobic compoenents. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yield vesicles that catalyzed light-dependent ATP formation. Both the 32Pi-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2'-dichloro-4'-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-beta-D-glucopyranosyl-4,6'-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

AFLP analysis of genetic polymorphism and evolutionary relationships among cultivated and wild Nicotiana species.

Amplified fragment length polymorphism (AFLP) analysis was used to determine the degree of intra- and inter-specific genetic variation in the genus Nicotiana. Forty-six lines of cultivated tobacco (Nicotiana tabacum L.) and seven wild Nicotiana species, including N. sylvestris, N. tomentosiformis, N. otophora, N. glutinosa, N. suaveolens, N. rustica, and N. longiflora, were analyzed, using at least eight different oligonucleotide primer combinations capable of detecting a minimum of 50 polymorphic bands per primer pair. The amount of genetic polymorphism present among cultivated tobacco lines (N. tabacum) was limited, as evidenced by the high degree of similarity in the AFLP profiles of cultivars collected worldwide. Six major clusters were found within cultivated tobacco that were primarily based upon geographic origin and manufacturing quality traits. A greater amount of genetic polymorphism exists among wild species of Nicotiana than among cultivated forms. Pairwise comparisons of the AFLP profiles of wild and cultivated Nicotiana species show that polymorphic bands present in N. tabacum can be found in at least one of three proposed wild progenitor species (i.e., N. sylvestris, N. tomentosiformis, and N. otophora). This observation provides additional support for these species contributing to the origin of N. tabacum.     

Origin of Nicotiana tabacum detected by primary structure of fraction I protein.

Nicotiana tabacum is believed to have arisen after hybridization of Nicotiana sylvestris with a species in the Tomentosae section of the genus Nicotiana. Recent biochemical experiments have confirmed the conclusions from previous cytogenetic studies that N. sylvestris was the maternal parent and have indicated that Nicotiana tomentosiformis was the paternal parent. However, these studies did not take into account the possibility that a new species of Nicotiana, called K-12, discovered in South America in 1968, could also have been one of the parents. Fraction I proteins were purified from N. tabacum and its putative progenitors, and separated into large and small subunits. Chymotryptic peptides of each subunit were analyzed by ion exchange column chromatography with a gradient elution system. Among 38 resolved peaks of the large subunits, 2 peaks were found to be different among the putative species. Since only N. sylvestris showed an identical chromatogram with N. tabacum, N. sylvestris was concluded to be the maternal progenitor, as the genetic information for the large subunit of Fraction I protein was known to be inherited by the cytoplasmic mode. On the other hand, the small subunit of Fraction I protein is inherited by the Mendelian mode and therefore N. tabacum, an allopolyploid, could be expected to contain two types of small subunits, one derived from N. sylvestris and the other from a paternal progenitor. N. sylvestris lacks two of the 25 chymotryptic peptides of the small subunit of N. tabacum. Among 3 putative paternal progenitors, these two peaks appeared only in N. tomentosiformis, but not in Nicotiana otophora or K-12. Thus, N. tomentosiformis was concluded to be a paternal progenitor of N. tabacum. The conclusion was verified by comparing chymotryptic peptides of small subunits from three amphidiploids of N. sylvestris crossed with N. tomentosiformis, N. sylvestris crossed with N. otophora snd N. sylvestris crossed with K-12. The analytical results showed that only the progeny of N. sylvestris crossed with N. tomentosiformis contained the same small subunits as N. tabacium.     

Inhibition by triphenyltin chloride of a tightly-bound membrane component involved in photophosphorylation.

At very low concentrations (less than 1 muM) triphenyltin chloride inhibits ATP formation and coupled electron transport in isolated spinach chloroplasts. Basal (-Pi) and uncoupled electron transport are not affected by triphenyltin. The membrane-bount ATP in equilibrium Pi exchange and Mg2+-dependent ATPase activities of chloroplasts are also completely sensitive to triphenyltin, although the Ca2+-dependent and Mg2+-dependent ATPase activities of the isolated coupling factor protein are insensitive to triphenyltin. The light-driven proton pump in chloroplasts is stimulated (up to 60%) by low levels of triphenyltin. Indeed, the amount of triphenyltin necessary to inhibit ATP formation or stimulate proton uptake is dependent upon the amount of chloroplasts present in the reaction mixture, with an apparent stoichiometry of 2-2.5 triphenyltin molecules/100 chlorophyll molecules at 50% inhibition of ATP formation and half-maximal stimulation of proton uptake. Chloroplasts partially stripped of coupling factor by an EDTA was are no longer able to accumulate protons in the light. However, low levels of triphenyltin can effectively restore this ability. The amount of triphenyltin required for the restoration of net proton uptake is also dependent upon the amount of chloroplasts, with a stoichiometry of 4-5 triphenyltin molecules/100 chlorophyll molecules at 50% reconstitution. On the basis of this and other evidence it is concluded that triphenyltin chloride inhibits phosphorylation.Atp in equilibrium Pi exchange and membrane-bound ATPase activities in chloroplasts by specifically blocking the transport of protons through a membrane-bound carrier or channel located in a hydrophobic region of the membrane at or near the functional binding site for the coupling factor.