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.     

Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids)

A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids.     

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.     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.     

AMP photophosphorylation and binding by chloroplasts

Stoichiometric amounts of chloroplast thylakoids photophosphorylate free AMP to tightly bound ADP. Free ADP is a poor competitor for this AMP photoreaction, which saturates below 16 micronAMP. The inhibitor, diadenosine pentaphosphate, abolishes AMP photophosphorylation, and inhibits dark ADP binding. Taken together, these data imply that this photoreaction involves the high affinity nucleotide binding site(s) of chloroplast coupling factor CF1, and that little mixing with free nucleotides occurs.     

Function of tightly bound nucleotides on membrane-bound chloroplast coupling factor

The kinetic behavior of tightly bound nucleotides on chloroplast coupling factor from spinach was determined under phosphorylating and nonphosphorylating conditions. Chloroplast coupling factor 1 (CF1) was labeled with tightly bound radioactive ADP and/or ATP at two specific sites and reconstituted with thylakoid membranes depleted of CF1 by treatment with NaBr. The initial incorporation and dissociation of ADP from one of the sites requires light but occurs at the same rate under phosphorylating and non-phosphorylating conditions. The initial rate is considerably slower than the rate of ATP synthesis, but nucleotide exchange is very rapid during steady-state ATP synthesis. A direct correspondence between this nucleotide binding site and a site on soluble CF1 that hydrolyzes ATP was demonstrated. A second site binds MgATP very tightly; the MgATP does not dissociate during ATP synthesis nor does its presence alter the rate of ATP synthesis. This is analogous to the behavior found for soluble CF1 during ATP hydrolysis. These results demonstrate that the tight-binding nucleotide sites on soluble CF1 and membrane-bound coupling factor are essentially identical in terms of binding properties and kinetic behavior during ATP hydrolysis and synthesis.     

Important subunit interactions in the chloroplast ATP synthase

General structural features of the chloroplast ATP synthase are summarized highlighting differences between the chloroplast enzyme and other ATP synthases. Much of the review is focused on the important interactions between the epsilon and gamma subunits of the chloroplast coupling factor 1 (CF(1)) which are involved in regulating the ATP hydrolytic activity of the enzyme and also in transferring energy from the membrane segment, chloroplast coupling factor 0 (CF(0)), to the catalytic sites on CF(1). A simple model is presented which summarizes properties of three known states of activation of the membrane-bound form of CF(1). The three states can be explained in terms of three different bound conformational states of the epsilon subunit. One of the three states, the fully active state, is only found in the membrane-bound form of CF(1). The lack of this state in the isolated form of CF(1), together with the confirmed presence of permanent asymmetry among the alpha, beta and gamma subunits of isolated CF(1), indicate that ATP hydrolysis by isolated CF(1) may involve only two of the three potential catalytic sites on the enzyme. Thus isolated CF(1) may be different from other F(1) enzymes in that it only operates on 'two cylinders' whereby the gamma subunit does not rotate through a full 360 degrees during the catalytic cycle. On the membrane in the presence of a light-induced proton gradient the enzyme assumes a conformation which may involve all three catalytic sites and a full 360 degrees rotation of gamma during catalysis.     

Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme.     

Cross-reconstitution of the F0F1-ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit delta

F0F1-ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton. The delta subunit of F1 plays an important role at the interface between the channel portion F0 and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CF0 and in Escherichia coli it is required for binding of EF1 to EF0. We wanted to know whether or not delta of one species was effective between F0 and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1(-delta), and delta were added in various combinations to the F1-depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate-mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching. Addition of CF1 to partially CF1-depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(-delta)+chloroplast delta, EF1, EF1(-delta)+E. coli delta were also effective but to lesser extent. CF1(-delta)+E. coli delta and EF1(-delta)+chloroplast delta restored photophosphorylation to a small but still significant extent. With F1-depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1 (-delta)+chloroplast delta and CF1(-delta)+E. coli delta gave approximately half the extent of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching as compared to EF1 or EF1(-delta)+E. coli delta by energization of the vesicles with NADH, while Ef1(-delta)+chloroplast delta was ineffective. All 'mixed' combinations were probably reconstitutively active only by plugging the protonic leak through the exposed F0 (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross-reconstitution is stunning in view of the weak sequence similarity between chloroplast delta and E. coli delta. It favors a role of delta as a conformational transducer rather than as a proton conductor between F0 and F1.     

The proton channel, CF0, in thylakoid membranes. Only a low proportion of CF1-lacking CF0 is active with a high unit conductance (169 fS)

We investigated the conductance of pea thylakoid membranes and their capacity for photophosphorylation as function of the extraction of chloroplast coupling factor CF1. The degree of extraction was varied via the incubation time in EDTA-containing hypo-osmolar medium and was measured by rocket electroimmunodiffusion. The conductance of thylakoid membranes was measured by flash kinetic spectrophotometry. The time course of extraction followed the time course of thylakoid swelling. Contrary to expectation increasing loss of CF1 did not primarily increase the velocity of proton efflux from each vesicle. Instead proton-tight vesicles were converted to leaky ones, which lost phosphorylating activity. Two subpopulations occurred, although both types of vesicles, leaky and proton-tight ones, were CF1-depleted to a similar degree. This implied that only a small fraction of CF1-lacking CF0 was functional as a proton channel. Tight vesicles had no functional channels while leaky ones had at least one. We determined the proportion of tight vesicles in three independent ways: via the residual phosphorylation activity, via measurements of proton efflux and via measurements of the electric relaxation across the membrane. The results obtained were identical. A statistical evaluation of the data led us to the following conclusions. EDTA treatment produced vesicles containing approximately 10(5) chlorophyll molecules, equivalent to a total of approximately 100 CF0CF1 per vesicle. Even at the highest degree of extraction (75% of total CF1 extracted) only 2.5 out of 75 exposed CF0 per vesicle were proton-conducting. The unit conductance of one open CF0 channel was 169 +/- 18 fS at pH 7.5 and room temperature. At an electrical driving force of 100 mV this was equivalent to the passage of approximately 10(5) protons/s. The most important consequence of this relatively high unit conductance was that a single open CF0 channel was capable of dissipating the protonmotive force of one vesicle, thereby deactivating the whole remaining catalytic capacity of this vesicle.     

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.     

Specific binding of coupling factor 1 lacking the delta and epsilon subunits to thylakoids

An improved procedure for the preparation of chloroplast coupling factor 1 (CF1) lacking the delta subunit is described. In addition, CF1 deficient in the epsilon subunit was isolated by a new method and CF1 lacking both of the smaller subunits was prepared. The ability of the subunit-deficient forms and of CF1, either heated or incubated with dithiothreitol to activate its ATPase activity, to bind to thylakoids from which CF1 had been removed was studied. All CF1 preparations bound in a cation-dependent manner to similar extents. CF1 lacking the delta subunit required higher cation concentrations for maximal binding. All preparations competed similarly with control CF1 for binding sites on the depleted membranes. The alpha subunit of all forms of CF1 in solution was rapidly cleaved by trypsin. After reconstitution, however, the alpha subunit of CF1, as well as of the subunit-deficient and the activated forms, was resistant to attack by trypsin. Moreover, treatment of the membranes with either trypsin or N,N'-dicyclohexylcarbodiimide inhibited the binding of all CF1 forms. These results suggest that the binding of the subunit-deficient and activated forms of CF1 is specific. CF1 lacking the epsilon subunit restored neither proton uptake nor ATP synthesis to the depleted membranes. In contrast to our previous results, CF1 lacking the delta subunit was partially effective. Previously, we used a suboptimal Mg2+ concentration for binding the delta-deficient enzyme which we show here was partially deficient in the epsilon subunit. These results show that the delta and epsilon subunits are not required for binding CF1 to the membranes and that the delta subunit is not an absolute requirement for ATP synthesis.     

Interactions of inorganic phosphate with spinach coupling factor 1. Effects on ATPase and ADP binding activities

Illumination of chloroplast thylakoid membranes results in both the release of adenine nucleotides from the tight nucleotide binding site(s) on chloroplast coupling factor 1 (CF1) and the activation of a light-triggered ATPase activity of CF1. Because inorganic phosphate stabilizes the light-triggered ATPase activity of CF1 in the dark, the effects of Pi on the rebinding of ADP to CF1 and on the light-triggered ATPase activity have been studied. Pi appears to be a partial noncompetitive inhibitor, with respect to ADP, of adenine nucleotide binding to the tight nucleotide binding site(s) on CF1 and induces negative cooperativity. The latter result suggests the existence of heterogeneous ADP binding sites in the presence of Pi. However, even under conditions where Pi causes a 50% reduction of tightly bound ADP, the ADP-induced dark decay of the ATPase activity is still complete. It was found that Pi inhibition of the light-induced dark binding of ADP can be reversed by the removal of the Pi. Removal of Pi also induces a small but significant ATPase activity. A model for the roles of the adenine nucleotide tight binding site(s) and Pi in the modulation of the spinach CF1 ATPase activity is proposed.     

Reactions of fluorescent probes with normal and chemically modified myelin basic protein and proteolipid. Comparisons with myelin.

Basic (encephalitogenic) protein and water-soluble proteolipid apoprotein isolated from bovine brain myelin bind 8-anilino-1-naphthalenesulfonate and 2-p-toluidinylnaphthalene-6-sulfonate with resulting enhancement of dye fluorescence and a blue-shift of the emission spectrum. The dyes had a higher affinity and quantum yield, when bound to the proteolipid (Kans=2.3x10--6,=0.67) than to the basic protein (Kans=3.3x10--5,=0.40). From the efficiency of radiationless energy transfer from trytophan to bound ANS the intramolecular distances were calculated to be 17 and 27 A for the proteolipid and basic protein, respectively. Unlike myelin, incubation with proteolytic enzymes (e.g., Pronase and trypsin) abolished fluorescence enhancement of ANS or TNS by the extracted proteins. In contrast to myelin, the fluorescence of solutions of fluorescent probes plus proteolipid was reduced by Ca-2+,not affected by La-3+, local anesthetics, or polymyxin B, and only slightly increased by low pH or blockade of free carboxyl groups. The reactions of the basic protein were similar under these conditions except for a two- to threefold increase in dye binding in the presence of La-3+, or after blockade of carboxyl groups. N-Bromosuccinimide oxidation of tryptophan groups nearly abolished native protein fluorescence, but did not affect dye binding. However, alkylation of tryptophan groups of both proteins by 2-hydroxy (or methoxy)-5-nitrobenzyl bromide reduced the of bound ANS (excited at 380 nm) to 0.15 normal. The same effect was observed with human serum albumin. The fluorescence emission of ANS bound to myelin was not affected by alkylation of membrane tryptophan groups with the Koshland reagents, except for abolition of energy transfer from tryptophan to bound dye molecules. This suggests that dye binding to protein is negligible in the intact membrane. Proteolipid incorporated into lipid vesicles containing phosphatidylserine did not bind ANS or TNS unless Ca-2+, La-3+, polymyxin B, or local anesthetics were added to reduce the net negative surface potential of the lipid membranes. However, binding to protein in the lipid-protein vesicles remained less than for soluble protein. Basic protein or bovine serum albumin dye binding sites remained accessible after equilibration of these proteins with the same lipid vesicles. It is proposed that in the intact myelin membrane the proteolipid is probably strongly associated with specific anionic membrane lipids (i.e., phosphatidylserine), and most likely deeply embedded within the lipid hydrocarbon matrix of the myelin membrane. Also, in the intact myelin membrane the fluorescent probes are associated primarily, if not solely with the membrane lipids as indicated by the binding data. This is particularly the case for TNS where the total number of myelin binding sites is three to four times the potential protein binding sites.     

Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor, CF1.

Effects of adenylates on chloroplast delayed light emission, at millisecond dark times, are inverse to the previously characterized effects of adenylates on electron transport rates. Either ADP alone or ATP alone increase intensity of delayed light, while ADP plus Pi decrease it. ADP alone requires the presence of an electron acceptor to have this effect on delayed light, but ATP does not. All three adenylate effects are abolished by uncoupling with gramicidin, by partial removal of photophosphorylation coupling factor (CF1) with EDTA, and by antibody to CF1. Readdition of CF1 re-established the adenylate effects in EDTA-stripped membranes. The three adenylate effects are differentially sensitive to pH, and pH differentially affected their abolition by antibody to CF1. The two adenylate effects shown in the absence of Pi are exhibited at lower adenylate concentrations than the ADP plus Pi effect, and are also less sensitive to phloridzin. These results are discussed in terms of probable adenylate effects on membrane-bound chloroplast coupling factor, CF1. At least two ADP binding sites would differ with respect to adenylate concentration for half maximal binding; pH of optimal binding capacity; phloridzin sensitivity; and functional regulation of electron transport, proton uptake, and energy storage within the membrane as measured by delayed light emission. It remains unclear whether the high affinity ADP binding site is identical to a high affinity ATP binding site on CF1.     

Reconstitution of photophosphorylation in EDTA-treated thylakoids by added chloroplast coupling factor 1 (ATPase) and chloroplast coupling factor 1 lacking the delta subunit. Structural or functional?

Upon EDTA treatment thylakoids lose the chloroplast coupling factor 1 (CF1) part of their ATP synthase, CF0CF1, this exposes the proton channel, CF0. The previously established ability of the CF1 subunit delta to block the proton leak through CF0 prompted us to study (a) the ability of complete CF1 and, for comparison, CF1 lacking the delta subunit to block proton leakage and thereby to reconstitute structurally some photophosphorylation activity of the remaining CF0CF1 molecules and (b) their ability to form functional enzymes (functional reconstitution). In order to discriminate between activities caused by added CF1 or CF1(-delta) and remaining CF0CF1, the former were inhibited by chemical modification of subunit beta by N,N'-dicyclohexyl carbodiimide (DCCD) and the latter by tentoxin. We found that added CF1 acted both structurally and functionally while added DCCD-treated CF1 (DCCD-CF1) acted only structurally. In contrast to previous observations, CF1(-delta) and DCCD-CF1(-delta) also acted structurally although the reduction of proton leakage was smaller than with DCCD-CF1. Hence there was no functional reconstitution without subunit delta present. Previous studies indicated that only a small fraction of exposed CF0 is highly conducting and that this small fraction is distinguished by its high affinity for added CF1. The results of this study point rather to a wider distribution of CF0 conductance states and binding affinities.     

Covalent binding of 1,N(6)-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP-synthase

The catalytic and noncatalytic sites of the chloroplast coupling factor (CF1) were selectively modified by incubation with the dialdehyde derivative of the fluorescent adenosine diphosphate analogue 1,N(6)-ethenoadenosine diphosphate. The modified CF1 was reconstituted with EDTA-treated chloroplast thylakoid membranes. The influence of light-induced transmembrane proton gradient and of phosphate ions on the fluorescence of 1,N(6)-ethenoadenosine diphosphate covalently bound to catalytic sites of reconstituted CF1 (ATP-synthase) was studied. Upon illumination of thylakoid membranes with saturating white light, the quenching of fluorescence of covalently bound 1,N(6)-ethenoadenosine diphosphate was observed. The quenching was reversed by the addition of inorganic phosphate to the reaction mixture in the dark. Repeated illumination induced the quenching once again: however, the addition of phosphate ions did not affect the fluorescence intensity now. When 1,N(6)-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP-synthase, no similar fluorescent changes were observed. The interrelation of the observed changes of 1,N(6)-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent changes in the structure of the catalytic site of ATP-synthase is discussed.     

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.     

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.     

Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle

Plastocyanin and chloroplast coupling factor 1 (CF(1)) are released from spinach (Spinacia oleracea L.) thylakoids during a slow freezethaw cycle. CF(1) addition increases the proton uptake of thylakoids previously frozen in sucrose concentrations of 15 mm to 100 mm. Addition of CF(1) and plastocyanin restores the proton uptake of thylakoids frozen in 100 mm sucrose. Plastocyanin and CF(1) release is a manifestation, not the cause, of freeze-thaw damage.Frozen-thawed thylakoids appear to exhibit two levels of response to sucrose as measured by light-dependent proton uptake. Different levels of protection afforded by sucrose may be due, in part, to quantitative differences in CF(1) release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plastocyanin release, CF(1) release, and disruption of the semi-permeability of thylakoids.