Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material.The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic cross-point (pH 4.1) was observed for the fraction enriched in inside-out grana thylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

EPR signals and orientation of cytochromes in the spinach chloroplast thylakoid membrane

The cytochromes in spinach chloroplasts were studied using EPR spectroscopy. In addition to the low-spin heme signals previously assigned, cytochrome f (g_z 3.51), high-potential cytochrome b-559 (g_z 3.08) and cytochrome b-559 converted to a low-potential form (g_z 2.94), a high-spin heme signal was induced by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). However, this signal cannot be due to cytochrome b-563 in its native form. The orientation of the cytochromes in the thylakoid membrane was studied in magnetically oriented chloroplasts. Cytochrome b-559 in the native state and in the low-potential form was found to have its heme plane perpendicular to the membrane plane. The orientation was the same for cytochrome b-559 oxidized by low-temperature illumination, which suggests that also the reduced heme is oriented perpendicular to the membrane.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Lipid-associated chlorophyll Evidence from 13C-NMR of the photosynthetic spinach thylakoid membrane

The ¹³C-NMR spectrum at 90.5 MHz has been obtained for the photosynthetic thylakoid membrane of spinach. Specific lipid and chlorophyll resonances can be assigned in the high resolution spectrum, although protein resonances are not observed. It can be estimated from resonance intensities that at least 30% of the plant chlorophyll contributes to the high resolution ¹³C spectrum with the remainder broadened by incomplete motional averaging. The resonance linewidths of the observed chlorophyll phytol chains are approximately the same as those of the lipid hydrocarbon chains, indicating a similar motional state and suggesting that this particular pool of chlorophyll is lipidbound or at most only loosely associated with proteins.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition.

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material. The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic crosspoint (pH 4.1) was observed for the fraction enriched in inside-out granathylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

Spin-label studies of the lipid regions of spinach thylakoids and a detergent-derived oxygen-evolving Photosystem II preparation

We have compared the fluidity of thylakoid membranes with the membrane present in a Triton X-100-derived, oxygen-evolving Photosystem II (PS II) preparation using two different spin labels. Data obtained with 2,2,6,6-tetramethylpipiridine-N-oxyl (TEMPO) shows that the PS II preparation contains less fluid membrane than the thylakoid. The TEMPO partition parameter (f) is about 2.5-times greater for the thylakoids at 6 mg chlorophyll/ml than for the PS II preparation at the same chlorophyll concentration. Similarly, the rotational correlation time, τ, of TEMPO residing in the membrane of the PS II preparation is about 2-times longer than the τ for TEMPO in the thylakoid membrane. A spin label which partitions more completely into the bilayer, 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl (7N14), indicates a much greater fluidity in the thylakoid membrane than the membrane of the PS II preparation. The PS II preparation appears to have a hydrocarbon phase which approaches the rigid limit of EPR detectable motion. These results are discussed in terms of possible lipid depletion in the PS II preparation and in terms of lateral heterogeneity of hydrocarbon fluidity in the thylakoid membrane caused by the lateral heterogeneity in protein components.     

The differential effects of short-time glutaraldehyde treatments on light-induced thylakoid membrane conformational changes, proton pumping and electron transport properties

A rapid quench technique utilizing the addition of excess buffer containing free amine groups (Tris, glycylglycine) to the reaction medium has enabled a detailed study of the time-course of glutaraldehyde inactivation on the spinach thylakoid membrane to be undertaken. The following light-induced parameters were inactivated in the sequence: slow transmittance changes (0–5 s) > coupling factor activity (5–20 s) > narrow angle 90° scattering changes (30–60 s). About 20% of PS II activity was lost by this treatment. No effect on activity, proton pumping and proton gradient formation was observed over the time-course studied. A consideration of these effects led to the proposal that the slow, light-induced transmittance changes reflect reversible thylakoid structural changes (unstacking, membrane flattening) in response to electron transport and the consequent proton pumping. The narrow angle 90° scattering changes were considered to reflect directly microconformational structural changes in response to the light-driven proton translocation as previously concluded from other workers.     

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q_B-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q_A (possibly also at the Q_B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.     

Isoelectric focusing and isoelectric points of aminoacyl-tRNA synthetases

Isoelectric points and isoelectric focusing behaviour of 10 highly purified eukaryotic aminoacyl-tRNA synthetases from 3 sources, Saccharomyces cerevisiae, Euglena gracilis and Phaseolus vulgaris were examined. The pI-values measured on polyacrylamide gels under native conditions are situated between pH 5.0-7.5. A microheterogeneity was observed for 9 enzymes appearing otherwise homogeneous on gel electrophoresis. A compilation of the isoelectric points of aminoacyl-tRNA synthetases is given and literature data are compared with our experimental results.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

Conversion of everted thylakoids into vesicles of normal sidedness exposing the outer grana partition membrane surface

Inside-out spinach thylakoid vesicles can be isolated by aqueous polymer two-phase partition following mechanical disruption of spinach chloroplast lamellae (Andersson, B and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472) and a mechanism for their formation has been experimentally supported (Andersson B., Sundby, C. and Albertsson, P.-Å. (1980) Biochim. Biophys. Acta 599, 391–402). Upon disruption, inside-out vesicles may form under stacking conditions, e.g., in 5 mM MgCl₂ or 150 mM NaCl, while disruption under destacking conditions, i.e., low concentrations of monovalent cations, gives only right-side-out vesicles. This study deals with the sidedness stability of the isolated inside-out thylakoid vesicles when stored or disrupted by sonication in various ionic environments. The sidedness of thylakoid vesicles was determined by their partition behaviour in an aqueous polymer phase system, direction of proton translocation and aggregation response (stacking) upon addition of MgCl₂. The results show that no spontaneous change from everted to normal sidedness occurs upon storage of the inside-out thylakoids. In contrast, sonication of these vesicles under destacking conditions (5 mM NaCl) results in a nearly complete transformation to right-side-out orientation. Also, in the presence of 5 mM MgCl₂ or 150 mM NaCl, sonication induced a change in sidedness of the inside-out vesicles but to a lesser extent. The stabilizing effect on the everted sidedness by cations was shown to be a result of preventing vesicle fragmentation by maintaining internal thylakoid appresions rather than by influencing the membrane curvature during resealing. Once released from an appressed state by overcoming the stacking forces, an opened thylakoid membrane shows an absolute preference for turning right-side-out in all media tested. These results strongly support the proposed formation mechanism, in which pairs of neighbouring grana membranes after disruption reseal with each other promoted by their close proximity. Since the inside-out vesicles derive from the grana appressions, their transformation back to normal sidedness exposes the outer membrane surface of appressed thylakoids. This region of the thylakoid membrane is normally hidden in the grana appressions and removal of grana leads concomitantly to lateral intermixing with non-appressed thylakoid components. Thus the current isolation of right-sided vesicles derived from the grana appressions should be a new tool for studies on the molecular organization of the thylakoid membrane.     

Regulation of photosynthesis in isolated spinach chloroplasts during orthophosphate limitation

The stromal concentration of orthophosphate in intact spinach chloroplasts (prepared in the absence of orthophosphate or pyrophosphate but supplied with both in the reaction medium) fell from a value of approx. 20 mM in the dark to a steady-state concentration of approx. 8 mM in the light. Chloroplasts illuminated in the absence of orthophosphate or pyrophosphate showed a similar trend. However, in this situation the stromal inorganic phosphate (P_i) concentration rapidly decreased from approx. 10 mM in the dark to a constant steady-state concentration of between 1.5 and 2.5 mM in the light. This P_i concentration was not further diminished (even though CO₂-dependent O₂ evolution had ceased) and was therefore considered to be stromal orthophosphate not freely available to metabolism. In the P_i-deficient chloroplasts the rate of photosynthesis declined rapidly after 1–2 min in the light such that CO₂-dependent O₂ evolution ceased with 5 min of the onset of illumination. The decline in O₂ evolution was accompanied by an increase in the transthylakoid ΔpH (as measured by 9-aminoacridine fluorescence quenching) and in the high-energy state, non-photochemical component of chlorophyll fluorescence quenching (q_E). Measurements of stromal metabolite concentrations showed that the ATP/ADP ratio was decreased in the P_i-deficient chloroplasts relative to chloroplasts illuminated in the presence of P_i. The stromal concentration of glycerate 3-phosphate was comparable in the P_i-deficient chloroplasts and those to which P_i had been supplied. Chloroplasts which were illuminated in P_i-free media showed a large accumulation of ribulose-1,5-bisphosphate relative to those supplied with P_i, suggesting inhibition of ribulose-1,5-bisphosphate carboxylase under these conditions. When P_i was added to chloroplasts illuminated in the absence of P_i, both non-photochemical quenching (q_E), photochemical quenching (q_Q) and ΔpH increased. This suggests that electron transport was not limited by inability to discharge transthylakoid ΔpH. These observation are consistent with the hypothesis that P_i limitation results in decreased ATP production by the thylakoid ATP synthase. The data presented here show that there are multiple sites of flux control exerted by low stromal P_i in the chloroplast. At least three factors contribute to the inhibition of photosynthesis under phosphate limitation: (1) there appears to be a direct effect of P_i on the energy-transducing system; (2) there is direct inhibition of the Calvin cycle decreasing the ability of the pathway to act as a sink for ATP and NADPH; and (3) feedback inhibition of primary processes occurs either via ΔpH or the redox state of electron carriers. However, ΔpH does not appear to be a limiting factor, but rather an inability to regenerate NADP as electron acceptor is suggested. The addition of DCMU to chloroplasts during illumination in the absence of P_i for periods of up to 10 min showed that there was very little loss of variable fluorescence despite a 60% reduction in the capacity for O₂ evolution. This would suggest that photoinhibitory damage to Photosystem II was not the major cause of the inhibition of photosynthesis observed with low P_i.     

Separation and characterization of stroma and grana membranes — evidence for heterogeneity in antenna size of both Photosystem I and Photosystem II

A rapid procedure to fractionate the thylakoid membrane into two well-separated vesicle populations, one originating from the grana and the other from the stroma-membrane region, has been developed. This was achieved by sonication of thylakoids present in an aqueous two-phase system followed by partitioning either by countercurrent distribution or by a batch procedure in three steps. The membrane populations were analysed according to their composition and photochemical activities. The grana membranes comprise, on chlorophyll basis, about 60% of the thylakoid material and are enriched in PS II, but also contain some PS I, while the stroma membranes comprise about 40% and are enriched in PS I, but also contain some PS II. Cytochrome f was slightly enriched in the grana-derived vesicle fraction. The properties of both PS I and PS II differ between the two populations. The PS I of the grana fraction (PS I) reached half-saturation at about half the light intensity of the PS I in the stroma-membrane fraction (PS I_β). The rate of P-700 photooxidation under low light illumination was higher for PS I than for PS I_β (30% larger rate constant), showing that PS I has a larger antenna. The PS II of the grana fraction (PS II) reached half-saturation at half the light intensity compared to the PS II of the stroma-membrane fraction (PS II_β). The results show that the grana-derived membranes contain PS I and PS II which have larger functional antenna sizes than the corresponding PS I_β and PS II_β of the stroma membranes. The results suggest that the photosystems of the grana are designed to allow effective electron transport both at low and high light intensities, while the stroma-membrane photosystems mainly work at high light intensities as a supplement to the grana systems.     

Determination of the cross-points of rat liver peroxisomes,peroxisomal core and the core components by cross-partition

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same crosspoint, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, d-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2:1. The theoretical value of cross-point of the core calculated from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

The influence of anions on oxygen evolution by isolated spinach chloroplasts

A study has been made of the onset of chloride deprivation on the oxygen-evolving characteristics of isolated spinach chloroplasts. Using a modulated oxygen electrode it is found that the type of inhibition depends on the anion replacing chloride in the bathing medium. With nitrate a large increase in phase lag accompanies a relatively small inhibition which can be shown to be consistent with a decrease in the rate constant of the reaction which limits the rate of electron transport between water and Photosystem II. With sulphate there is a very small phase change but a larger inhibition which suggests that replacing chloride with sulphate in an electron-transport chain shuts off that chain. With acetate there is a moderate increase in phase lag and the largest inhibitory effect. The phase-lag increase suggests that acetate is affecting the same chloride-sensitive site as nitrate. However, the inhibition cannot be explained by this effect alone and points to the existence of a second chloride-sensitive site. Of the four forward reactions associated with the Kok model of oxygen evolution (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) only S1₃ → S₀ is slowed down when chloride is replaced by nitrate. This reaction is not slowed down by replacing chloride with sulphate.     

Characterization of nucleotide binding sites on the membrane-bound chloroplast ATP synthase (coupling factor CF1)

Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF₁ are very fast reactions in the light, so that a direct comparison of both reactions is difficult. By adding substrate ADP and phosphate to illuminated thylakoids together with the uncoupler FCCP, the phosphorylation time is limited and the amount of ATP formed can be reduced to less than 1 ATP per enzyme. Low concentrations of medium nucleotides during illumination increase the amount of ATP formed during uncoupling presumably by binding to the tight nucleotide binding site (further designated as ‘site A’) with an affinity of 1 to 7 μM for ADP and ATP. ATP formation itself shows half-saturation at about 30 μM. Loosely bound nucleotides are exchanged upon addition of nucleotides with uncoupler (Schumann, J. (1984) Biochim. Biophys. Acta 766, 334–342). Release depends binding of nucleotides to a second site. The affinity of this site for ADP (in the presence of phosphate) is about 30 μM. It is assumed that phosphorylation and induction of exchange both occur on the same site (site B). During ATP hydrolysis, an ATP molecule is bound to site A, while on another site, ATP is hydrolyzed rapidly. The affinity of ADP for the catalytic site (70 μM) is in the same range as the observed Michaelis constant of ADP during phosphorylation; it is assumed that site B is involved in ATP hydrolysis. Site A exhibits some catalytic activity; it might be that site A is involved in ATP formation in a dual-site mechanism. For ATP hydrolysis, however, direct determination of exchange rates showed that the exchange rate of ATP bound to site A is about 30-times lower than ATP hydrolysis under the same conditions.     

Effects of cations on the adhesion between membrane vesicles obtained by digitonin fractionation of spinach chloroplasts

The heavy fraction obtained by digitonin treatment of stacked spinach chloroplasts, suspended in media with different ionic composition, was examined by electron microscopy. In the presence of 5 mM MgCl₂ the thylakoid fragments adhere to one another in a ‘stacked configuration,’ while, in the presence of 10 mM NaCl, mainly only single ‘unstacked’ vesicles are present, which, upon addition of 5 mM MgCl₂, completely revert to the stacked configuration. As previously reported (Chow, W.S. and Barber, J. (1980) Biochim. Biophys. Acta 593, 149–157), no difference in fractionation of chlorophyll between light and heavy fractions was seen after a second digitonin treatment of this fraction suspended in media containing different cation concentrations. From these results it was concluded: (1) that for the unstacking process the movement of proteins or complexes from the stromal to the granal lamellae is not required. Upon lowering the screening by cations of the surface negative charges, the membranes separate from one another; (2) that, under these conditions, as in others (Jennings, R.C., Gerola, P.D., Garlaschi, F.M. and Forti, G. (1980) FEBS Lett. 115, 39–42), digitonin fractionation is not a tool to investigate the degree of membrane stacking.     

Loose and tight binding of adenine nucleotides by membrane-associated chloroplast ATPase

Steady-state binding of adenine nucleotides by thylakoid membranes is measured by employing a centrifugation technique. By this method tightly bound nonexchangeable nucleotides can be discriminated from loosely bound, exchangeable nucleotides. Nucleotide binding requires membrane energization and is highly specific for medium ADP. In illuminated chloroplasts almost no exogenous AMP and only some ATP are incorporated, most being recovered as tightly bound nucleotides. In light-triggered chloroplasts, however, which are capable of hydrolyzing ATP, a high level of exchangeable nucleotides is found on the membranes. The sum of tightly bound and loosely bound nucleotides originating from medium ADP is about one per CF₁. The ratio between them decreases with increasing proton-motive force. Exchangeable nucleotides most probably represent the ligands involved in the catalytic process, as suggested from substrate specificity and the effect of a competitive inhibitor of photophosphorylation, naphthoyl ADP. This compound in a low concentration range supresses loose binding but not tight binding of medium ADP. Under phosphorylating conditions (presence of ADP, P_i and light), some of the tightly bound nucleotides exist as ATP even in the presence of a hexokinase system. The results are discussed in the context of the regulation of chloroplast ATPase by tight nucleotide binding.