Role of divalent cations and ascorbate in photochemical activitis of Anacystis membranes

Photosynthetic membrane fragments were prepared from Anacystic nidulans by French pressure cell disruption. Ascorbate was required to stabilize photophosphorylation activity in membranes kept at near 0 degrees C. Divalent cations were required during mechanical disruption and during assays for Photosystem II activity, with Ca2+ serving best. The rate of photophosphorylation was severely inhibited by Ca2+ during assays. Results suggest that best rates are achieved when photosynthetic membranes contain Ca2+ exposed to the interior surface, facilitating Photosystem II activity, and Mg2+ exposed to the exterior surface during assays, facilitating photophosphorylation activity.     

Increase effected by calcium ion in the rate of oxygen evolution from preparations of Phormidium luridum

The presence of Ca²⁺ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca²⁺ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl₂ in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca²⁺. This indicates that Ca²⁺ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca²⁺, discounting fatty acid mediation of the effects of Ca²⁺. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca²⁺.     

Cation-mediated regulation of excitation energy distribution in chloroplasts lacking organized Photosystem II complexes

Despite the total loss of Photosystem II activity, thylakoids isolated from the green nuclear maize mutant hcf1-3 contain normal amounts of the light-harvesting chlorophyll $\text{a}\text{b}$ pigment-protein complex (LHC). We interpret the spectroscopic and ultrastructural characteristics of these thylakoids to indicate that the LHC present in these membranes is not associated with Photosystem II reaction centers and thus exists in a ‘free’ state within the thylakoid membrane. In contrast, the LHC found in wild-type maize thylakoids shows the usual functional association with Photosystem II reaction centers. Several lines of evidence suggest that the free LHC found in thylakoids isolated from hcf1-3 is able to mediate cation-dependent changes in both thylakoid appression and energy distribution between the photosystems: (1) Thylakoids isolated from hcf1-3 and wild-type seedlings exhibit a similar Mg²⁺-dependent increase in the short/long wavelength fluorescence emission peak ratio at 77 K. This Mg²⁺ effect is lost following incubation of thylakoids isolated from either source with low concentrations of trypsin. Such treatment results in the partial proteolysis of the LHC in both membrane types. (2) Thylakoids isolated from both hcf1-3 and wild-type seedlings show a similar Mg²⁺ dependence for the enhancement of the maximal yield of room temperature fluorescence and light scattering; both Mg²⁺ effects are abolished by brief incubation of the thylakoids with low concentrations of trypsin (3) Mg²⁺ acts to reduce the relative quantum efficiency of Photosystem I-dependent electron transport at limiting 650 nm light in thylakoids isolated from hcf1-3. (4) The pattern of digitonin fractionation of thylakoid membranes, which is dependent upon structural membrane interactions and upon LHC in the thylakoids, is similar in thylakoids isolated from both hcf1-3 and wild-type seedlings. We conclude that the surface-exposed segment of the LHC, but not the LHC-Photosystem II core association, is necessary for the cation-dependent changes in both thylakoid appression and energy distribution between the two photosystems, and that the LHC itself is able to transfer excitation energy directly to Photosystem I in a Mg²⁺-dependent fashion in the absence of Photosystem II reaction centers. The latter phenomenon is equivalent to a cation-induced change in the absorptive cross-section of Photosystem I.     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Regulation of the Hill reaction by cations and its abolishment by uncouplers

Concurrent measurements of P-700 turnover and the reduction of K₃Fe(CN)₆ revealed an identical relative quantum yield for both reactions in isolated pea chloroplasts as well as chloroplast particles from wild type Scenedesmus. On the other hand, chloroplast particles of wild type Scenedesmus showed the same relative quantum yield for the Hill reaction as those of the P-700-free mutant No. 8, indicating that P-700 is not required for ferricyanide reduction.Several metal ions, such as Mg²⁺, Ca²⁺, Na⁺ and K⁺ stimulated the reduction of K₃Fe(CN)₆. In short wavelength light, the stimulation was a function of light intensity, varying in magnitude from an approximate doubling of the yield in low intensities to only a slight increase at light saturation. P-700 was totally unaffected by the cations.The effect of the metal salts was abolished in the presence of uncouplers of photophosphorylation.The data reconcile several divergent results concerning the effect of divalent cations on the reduction of ferricyanide which have been reported in the recent literature. On the whole the experiments suggest that the Hill acceptor can be reduced at two sites. The stimulation of the Hill reaction by metal ions is proposed to be due to an activation of Photosystem II and a more efficient utilization of quanta at the expense of radiationless de-excitation.     

On the nature of the fluorescence decrease due to phosphorylation of chloroplast membrane proteins

1. Phosphorylation of chloroplast membranes by illumination in the presence of ATP results in a 15–20% increase in the rate of Photosystem I electron transfer at low light intensity. 2. Phosphorylated membranes when depleted of Mg²⁺ and resuspended in a low salt medium still show a 17% lower yield of Photosystem II fluorescence than do unphosphorylated membranes. A 31% difference is seen after restoration of the maximal yield by addition of Mg²⁺. 3. The concentration of Mg²⁺ required to induce a half-maximal increase in fluorescence is 0.9 mM for control and 1.8 mM for phosphorylated chloroplasts. Phosphorylation at 1 mM Mg²⁺ can therefore cause more than double the amount of decrease in fluorescence yield from Photosystem II compared to phosphorylation at 5 mM. 4. The above results are discussed in terms of the mechanism of the ATP-induced fluorescence changes and a suggestion is made that the apparent interaction between phosphorylation and Mg²⁺ concentration may be a physiologically important phenomenon.     

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Reversible inhibition of photochemistry of photosystem II by Ca2+ removal from intact cells of Anacystis nidulans

Depletion of Ca²⁺ from Anacystis nidulans produces an inhibition of O₂ evolution that is accompanied both at 39°C and 77 K by a loss of chlorophyll fluorescence of variable yield. This indicates that Ca²⁺-depletion causes disruption of normal photosystem II function, manifested by the disappearance of photoreduction of Q. Delayed light emission in the ms time range is also eliminated in Ca²⁺-depleted cells, which confirms that Ca²⁺ removal prevents charge separation and recombination in reaction centers of photosystem II. Readdition of Ca²⁺ to depleted cells restores fully the fluorescence of variable yield and delayed light emission, as well as O₂ evolution. Thus, Ca²⁺ may be a required component for photosystem II in A. nidulans.     

Modification of the thermoluminescence properties of Ca2+ depleted Photosystem II membranes by the 23 kDa extrinsic polypeptide and by oligocarboxylic acids

Photosystem II membranes were isolated from chloroplasts of pokeweed (Phytolacca americana) and rendered deficient in Ca²⁺, an inorganic cofactor of photosynthetic water oxidation. The thermoluminescence properties of such membranes were found to depend on the Ca²⁺-depleting method used. This feature was analyzed with respect to the thermoluminescence emission that accompanied the recombination reaction between the reduced acceptor Q_A ^– and the oxidant of the S₂ state. It was determined that the differences observed among various preparations of Ca²⁺-depleted membranes were attributable to the presence or absence of the extrinsic 23 kDa polypeptide on the membranes. The binding of this polypeptide to Ca²⁺-depleted membranes devoid of the 17 and 23 kDa extrinsic polypeptides caused the thermoluminescence to be emitted at a higher temperature due to a further stabilization of an already abnormally stable S₂ state. Addition of the chelators EDTA or EGTA and of citrate brought about a similar response. The conditions required for the upshift of the emission temperature of thermoluminescence strongly resembled those identified by Boussac et al. (FEBS Lett. 277 (1990) 69–74) as responsible for modifying the EPR multiline signal from the S₂ state of Ca²⁺-depleted PS II membranes. Consistent with the authors' interpretation of the reason for this modification, we conclude that the elevated emission temperature of the thermoluminescence emission reflects an abnormal ligand environment of the Mn-center in PS II that may be created by a direct ligation of the added agents to Mn. Evidence is also presented that the return to a normal S₂ after an addition of Ca²⁺ occurs via yet another condition of S₂ which, in terms of its thermoluminescence properties, resembles that of Ca²⁺-depleted membranes before addition of modifying agents, but is not identical to it.     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. II. Evidence for surface localization of Photosystem II reaction centers

The enzyme lactoperoxidase was used to specifically iodinate the surface-exposed proteins of chloroplast lamellae. This treatment had two effects on Photosystem II activity. The first, occurring at low levels of iodination, resulted in a partial loss of the ability to reduce 2,6-dichlorophenolindophenol (DCIP), even in the presence of an electron donor for Photosystem II. There was a parallel loss of Photosystem II mediated variable yield fluorescence which could not be restored by dithionite treatment under anaerobic conditions. The same pattern of inhibition was observed in either glutaraldehyde-fixed or unfixed membranes. Analysis of the lifetime of fluorescence indicated that iodination changes the rate of deactivation of the excited state chlorophyll. We have concluded that iodination results in the introduction of iodine into the Photosystem II reaction center pigment-protein complex and thereby introduces a new quenching. The data indicate that the reaction center II is surface exposed.At higher levels of iodination, an inhibition of the electron transport reactions on the oxidizing side of Photosystem II was observed. That portion of the total rate of photoreduction of DCIP which was inhibited by this action could be restored by addition of an electron donor to Photosystem II. Loss of activity of the oxidizing side enzymes also resulted in a light-induced bleaching of chlorophyll a₆₈₀ and carotenoid pigments and a dampening of the sequence of O₂ evolution observed during flash irradiation of treated chloroplasts. All effects on electron transport on the oxidizing side of Photosystem II could be eliminated by glutaraldehyde fixation of the chloroplast lamellae prior to lactoperoxidase treatment. It is concluded that the electron carriers on the oxidizing side of Photosystem II are not surface localized; the functioning of these components is impaired by structural disorganization of the membrane occurring at high levels of iodination.Our data are in agreement with previously published schemes which suggest that Photosystem II mediated electron transport traverses the membrane.     

Molecular organization of oxygen-evolution system in chloroplast

The main function of Photosystem II in chloroplast is to oxidize water molecules to produce oxygen. Strong oxidant produced by photoreaction at Photosystem II reaction center derives electrons from water and the electrons are transferred via Photosystem I to NADP⁺. The components required for water oxidation in Photosystem II were identified and their molecular properties as well as their roles in the oxygen evolution process were elucidated. The entity of the oxygen evolution system is a supramolecular complex of Photosystem II in the thylakoid membrane where reaction center binding polypeptides, three extrinsic polypeptides, managenese atoms, Ca²⁺ and Cl⁻ ions are the essential components, and they constitute a specific catalytic domain for water oxidation. Recipient of the Botanical Society Award for Young Scientists, 1988.     

Calcium ions can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution

Photosystem II particles were prepared from spinach chloroplasts with Triton X-100, and treated with 1.0 M NaCl to remove polypeptides of 24 kDa and 18 kDa and to reduce the photosynthetic oxygen-evolution activity by about half. Oxygen-evolution activity was restored almost to the original level with 10 mM Ca²⁺, in a similar manner to the rebinding of 24-kDa polypeptide. Other cations such as magnesium, sodium and manganese ions could not restore any oxygen-evolution activity. These observations, together with a kinetic analysis, suggest that Ca²⁺ can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution in Photosystem II particles.     

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP⁺. Magnesium ions were required not only for light-induced electron transport from water to NADP⁺ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg²⁺ lost the capacity to photoreduce NADP⁺ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP⁺ by DCIPH₂ was independent of Mg²⁺ (or Cl^?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl₂ produced a strong inhibition of NADP⁺ photoreduction with DCIPH₂ without significantly affecting the rate of NADP⁺ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP⁺ with different electron donors are discussed.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Photooxidation of ferrocyanide and iodide ions and associated phosphorylation in NH2OH-treated chloroplasts

NH₂OH-treated, non-water oxidizing chloroplasts are shown to be capable of oxidizing ferrocyanide and I^? via Photosystem II at appreciable rates (? 200 μequiv/h per mg chlorophyll). Using methylviologen as electron acceptor, ferrocyanide oxidation can be measured as O₂ uptake, as ferricyanide formation, or as H⁺ consumption (2 Fe²⁺ + 2H⁺ + O₂ → 2 Fe³⁺ + H₂O₂). I^? oxidation can be measured as methylviologen-mediated O₂ uptake, or spectrophotometrically, using ferricyanide as electron acceptor. The oxidation product I₂ is re-reduced, as it is formed, by unknown reducing substances in the reaction system.The rate-saturating concentrations of these donors are very high: 30 mM with ferricyanide and 15 mM with I^?. Relatively lipophilic Photosystem II donors such as catechol, benzidine and p-aminophenol saturate the photooxidation rate at much lower concentrations (< 0.5 mM). It thus seems that the oxidation of hydrophilic reductants such as ferricyanide and I^? is limited by permeability barriers. Very likely the site of Photosystem II oxidation is embedded in the thylakoid membrane or is situated on the inner surface of the membrane.The efficiency of phosphorylation (P/e₂) is 0.5 to 0.6 with ferrocyanide and about 0.5 with I^?. In contrast the P/e₂ ratio is 1.0 to 1.2 when water, catechol, p-aminophenol or benzidine serves as electron donor. These differences imply that only one of two phosphorylation sites operate when ferrocyanide and I^? are oxidized. Ferrocyanide and I^? are also chemically distinct from other Photosystem II donors in that their oxidation does not involve proton release. It is suggested that the mechanism of energy conservation associated with Photosystem II may be only operative when the removal of electrons from the donor results in release of protons (i.e. with water, hydroquinones, phenylamines, etc.).     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Inactivation and Calcium-Dependent Reactivation of Oxygen Evolution in Photosystem II Preparations Treated at pH 3.0 or with High Concentrations of NaCl

A brief treatment at pH 3.0 of Photosystem II (PS II) membranescontaining two bound Ca²⁺ from rice resulted in strong suppressionof oxygen evolution concomitant with extraction of one Ca²⁺and the lost activity was restored on addition of 50 mM Ca²⁺.However, inactivation of oxygen evolution by low pH-treatmentof oxygen-evolving PS II complexes containing only one Ca²⁺from a rice chlorophyll b-deficient mutant was not associatedwith extraction of the bound Ca²⁺, although oxygen evolutionwas markedly enhanced by the addition of Ca²⁺ to the treatedcomplexes. Thus, the acid-inactivation of oxygen evolution cannotbe related to extraction of Ca²⁺. On the other hand, low pH-treatmentwas found to share the following common features with NaCl-treatmentwhich also causes a Ca²⁺-reversible inactivation of oxygen evolution.(1) Exposure of PS II membranes to pH 3.0 resulted in solubilizationof the 23 and 17 kDa extrinsic proteins, although the releasedproteins rebound to the membranes when pH was raised to 6.5.(2) There was an apparent heterogeneity in the binding affinityof Ca²⁺ effective in restoration of the oxygen-evolving activity.(3) Low pH-treated preparations required a higher concentrationof Ca²⁺ for the maximum reactivation of oxygen evolution thandid NaCl-washed preparations. This was also the case with Sr²⁺,which stimulated oxygen evolution of both low pH-treated andNaCl-washed PS II membranes to smaller extents. When the extrinsic23 and 17 kDa proteins had been removed, however, Ca²⁺ concentrationdependence of oxygen evolution in low pH-treated membranes becamesimilar to that in NaCl-washed PS II preparations and the changeswere largely reversed by rebinding of the two proteins. Theseresults strongly suggest that low pH-treatment and NaCl-washinvolve similar mechanisms of Ca²⁺-dependent reactivation. ¹ Present address: Solar Energy Research Group, The Instituteof Physical and Chemical Research (RIKEN), Wako, Saitama, 351-01Japan (Received August 27, 1990; Accepted February 12, 1991)     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Reversible shift between two states of Ca2+-ATPase in human erythrocytes mediated by Ca2+ and a membrane-bound activator

The (Ca²⁺ + Mg²⁺)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation.The A-state showed low maximum activity (V) and the Ca²⁺ activation was characterized by a Hill coefficient, n_H, of about 1 and a Michaelis constant, K_Ca, about 30 μM.The B-state showed high V, a n_H above 1, which indicates positive cooper-activity of Ca²⁺ activation, and a K_Ca of about 1 μM.With varying ATP concentrations, both the A-state and the B-state showed negative cooperativity and slightly different values of K_m.The B-state was shifted to the A-state when the membranes were exposed to low Ca²⁺ concentrations. The shift reached 50% at approx. 0.5 μM Ca²⁺. At the low Ca²⁺ concentrations an activator was released from the membranes.The A-state was shifted to the B-state when the membranes were exposed to Ca²⁺ in the presence of the activator. The shift reached 50% at about 30 μM Ca²⁺. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca²⁺ and activator accelerated the recovery.It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may represent a resting and an active state, respectively, of the calcium pump.