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⁺.     

Calcium and photosynthetic oxygen evolution in cyanobacteria.

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca(2+). Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca(2+) addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca(2+) and Mg(2+) stimulate turnover, unit activation is Ca(2+) specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca(2+) -activated preparation. The Ca(2+) requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca(2+) of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605--609) these observations strongly suggest a site of Ca(2+) action within Photosystem II. The pronounced specificity of the Ca(2+) requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca(2+) dependence in P. luridum, French-press treatment is required for maximal expression of Ca(2+) -specific effects. French-press breakage causes a release of endogenous Ca(2+) from cells, supporting the view that added Ca(2+) restores oxygen evolution by satisfying a physiological requirement for the cation.     

Isolation and characterization of oxygen-evolving photosystem II complexes retaining the PsbO, P and Q proteins from Euglena gracilis

Oxygen-evolving photosystem II (PSII) complexes of Euglena gracilis were isolated and characterized. (1) The PSII complexes contained three extrinsic proteins of 33 kDa (PsbO), 23 kDa (PsbP) and 17 kDa (PsbQ), and showed oxygen-evolving activity of around 700 micromol O2 (mg Chl)(-1) h(-1) even in the absence of Cl- and Ca2+ ions. (2) NaCl-treatment removed not only PsbP and PsbQ but also a part of PsbO from Euglena PSII, indicating that PsbO binds to Euglena PSII more loosely than those of other organisms. Treatments by urea/NaCl, alkaline Tris or CaCl2 completely removed the three extrinsic proteins from Euglena PSII. (3) Each of the Euglena extrinsic proteins bound directly to PSII independent of the other extrinsic proteins, which is similar to the binding properties of the extrinsic proteins in a green alga, Chlamydomonas reinhardtii. (4) One of the significant features of Euglena PSII is that the oxygen evolution was not enhanced by Ca2+. When CaCl2-treated Euglena PSII was reconstituted with PsbO, the oxygen-evolving activity was stimulated by the addition of NaCl, but no further stimulation was observed by CaCl2. (5) Oxygen evolution of Euglena PSII reconstituted with PsbO from C. reinhardtii or spinach instead of that from Euglena also showed no enhancement by Ca2+, whereas a significant enhancement of oxygen evolution was observed by Ca2+ when the green algal or higher plant PSII was reconstituted with Euglena PsbO instead of their own PsbO. These results indicate that the PSII intrinsic proteins instead of the extrinsic PsbO protein, are responsible for the stimulation of oxygen evolution by Ca2+. Sequence comparison of major PSII intrinsic proteins revealed that PsbI of Euglena PSII is remarkably different from other organisms in that Euglena PsbI possesses extra 16-17 residues exposed to the luminal side. This may be related to the loss of enhancement of oxygen evolution by Ca2+ ion.     

Photoreduction of Molecular Oxygen in Preparations of Photosystem II under Photoinhibitory Conditions

Preparations of photosystem II (PSII) from pea (Pisum sativum L.) leaves were used to study the evolution and reduction of molecular oxygen under photoinhibitory conditions. Under these conditions, the photoinduced oxygen uptake did not exceed 10% of the total oxygen-evolving activity in PSII preparations. Both the Hill and the Mehler reactions were found to occur simultaneously under long-term illumination of PSII preparations with high-intensity light in the presence of potassium ferricyanide. During this light treatment in the presence of potassium ferricyanide, the rate of oxygen uptake increased gradually reaching 30% of the oxygen-evolving activity. The photogeneration of superoxide anion radical at increasing light intensities followed a typical light-response curve with a light saturation at 800 W/m². The results provide evidence that the Mehler reaction is the major source for superoxide and hydrogen peroxide in PSII preparations under photoinhibitory conditions and that the Mehler reaction in PSII proceeds more effectively at high light intensities. The relatively low and sustained rate of oxygen photoreduction in PSII preparations under photoinhibitory conditions substantiates the hypothesis on the involvement of Mehler reaction in cell signaling and regulation.     

Calcium and photosynthetic oxygen evolution in cyanobacteria

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca²⁺. Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca²⁺ addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca²⁺ and Mg²⁺ stimulate turnover, unit activation is Ca²⁺ specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca²⁺-activated preparation.

The Ca²⁺ requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca²⁺ of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605–609) these observations strongly suggest a site of Ca²⁺ action within Photosystem II.

The pronounced specificity of the Ca²⁺ requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca²⁺ dependence in P. luridum, French-press treatment is required for maximal expression of Ca²⁺-specific effects. French-press breakage causes a release of endogenous Ca²⁺ from cells, supporting the view that added Ca²⁺ restores oxygen evolution by satisfying a physiological requirement for the cation.     

Enhancement by chloride ions of photoactivation of oxygen evolution in manganese-depleted photosystem II membranes

The Mn cluster that catalyzes photosynthetic oxygen evolution was removed from the photosystem II (PSII) complex by treating PSII membranes with 1.0 mM NH2OH with concomitant inactivation of oxygen evolution. The cluster was reconstituted by incubating the treated membranes with 1.0 mM Mn2+, 20 mM Ca2+, 10 microM 2,6-dichlorophenolindophenol, and Cl- under illumination with continuous or flashing light to restore the oxygen-evolving capacity. This light-dependent activation (photoactivation) of oxygen evolution did not occur to a significant extent at 3 mM Cl-, but markedly accelerated at higher Cl- concentrations without showing a saturation phenomenon even at 1 M Cl-. At 10 mM Cl- only about 10% of the oxygen-evolving activity before NH2OH treatment was restored by 5-min illumination with continuous light, whereas at 600 mM Cl- about 60% of the original activity was recovered. This acceleration resulted from at least two different actions of Cl-: (1) stabilization of the intermediate state involved in the photoactivation process and (2) increase in the quantum yield of photoactivation. The stabilization of the intermediate was saturated at about 150 mM Cl-, whereas the increase in yield did not show saturation. The Cl(-)-induced increase in quantum yield did not involve any changes in the affinity of either Mn2+ binding or Ca2+ binding for photoactivation, but was rather ascribed to a protective effect of Cl- against inhibition of photoactivation by high concentrations of Mn2+. We also found that removal of the extrinsic 33-kDa protein from the PSII complex increased the Cl- requirement for photoactivation.     

Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II.

Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence-quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition.     

The maximal velocity and the calcium affinity of the red cell calcium pump may be regulated independently

The kinetics of active Ca2+ transport in inside-out red cell membrane vesicles and the Ca2+-ATPase activity of the purified Ca2+ pump were studied and the effects of calmodulin, acidic phospholipids, and controlled trypsinization were compared. In the presence of calmodulin the maximal rate and the apparent affinity of the pump for Ca2+ were greatly increased in both preparations. The lowest value of Km(Ca) was between 0.5 and 0.7 microM depending on the concentration of calmodulin and on the enzyme preparation. Positive cooperativity for Ca2+ activation with a Hill coefficient of 1.6-1.7 was observed in all cases. When acidic phospholipids (phosphatidylinositol 4-phosphate was routinely used) were added to the inside-out vesicles or to the purified enzyme, maximal transport rates equal to those obtained with calmodulin were measured but the Km(Ca) decreased to 0.25 microM and the positive cooperativity disappeared (the Hill coefficient approached 1). Highly active, calmodulin-independent proteolytic fragments of molecular mass of 81 and 76 kDa were produced with controlled trypsinization. When the trypsin treatment was directed to obtain primarily the 81-kDa fragment, the preparation showed characteristics similar to those of the intact Ca2+ pump in the presence of calmodulin; that is, the same Vmax was obtained, the Km(Ca2+) was 0.5-0.6 microM, and the Hill coefficient was about 1.6. Addition of phosphatidylinositol 4-phosphate or allowing further proteolysis to produce the 76-kDa fragment, shifted the Km(Ca) to 0.25 and reduced the Hill coefficient to 1, without changes in the maximal rate. Based on these results it is suggested that the maximal velocity and the Ca2+ affinity on the erythrocyte Ca2+ pump may be regulated independently and that independent polypeptide regions of the enzyme are involved in the regulations.     

Oxygen evolution in the absence of the 33-kilodalton manganese-stabilizing protein.

There has been a considerable amount of controversy concerning the ability of photosystem II to evolve oxygen in the absence of the 33-kDa, manganese-stabilizing protein. Early reports indicated that some capacity for oxygen evolution existed in manganese-stabilizing protein-depleted membranes while more recent studies have suggested that the observed oxygen evolution activity arose from residual manganese-stabilizing protein present in the salt-washed preparations. In this paper, it is conclusively demonstrated that significant rates of steady-state oxygen evolution are observed in oxygen-evolving photosystem II membranes in the absence of detectable quantities of the manganese-stabilizing protein. More then 99% of the manganese-stabilizing protein was removed by either one CaCl2 or two NaCl-urea washes. The amount of manganese-stabilizing protein removed was quantified immunologically using mouse polyclonal antibodies. Oxygen evolution rates of 115-140 mumol of O2 (mg of Chl)-1 h-1 were observed in the NaCl-urea-washed preparations. These rates represent about 24% of the rate observed in untreated membranes [450-600 mumol of O2 (mg of Chl)-1 h-1]. Somewhat lower, although still significant rates were observed in the CaCl2-washed preparations. Optimal rates of oxygen-evolving activity in NaCl-urea-washed membranes which are devoid of the manganese-stabilizing protein required high concentrations of calcium and chloride.     

The effects of storage of sarcoplasmic reticulum fragments on the Ca2+, Mg2+-ATPase.

The effects of K+ and Na+ on the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum fragments (SRF) were investigated at 1 mM ATP. There was an alteration of the sensitivity of the ATPase to the monovalent cations during storage of the SRF preparation. The Ca2+, Mg2+-ATPase of freshly prepared SRF was slightly activated by 5-10 mM K+ and Na+. Mg2+-ATPase was inhibited by both the monovalent cations to the same extent, and this response to the ions was independent of the freshness of the preparations. After storage of SRF, however, the Ca2+,Mg2+-ATPase was markedly activated by higher concentrations of K+ and Na+ (0.2-0.3 M). K+ and Na+ reduced the Ca uptake at the steady state in freshly prepared SRF, but did not affect pre-steady state uptake. In the presence of oxalate, the rate of Ca accumulation both in fresh and stored preparations was activated by 0.1-0.2 M K+ and Na+. The Ca2+, mg2+-ATPase with oxalate, so-called "extra ATPase," showed the same response to the ions as did the activity without oxalate during storage.     

A high-potential redox component located within cyanobacterial photosystem II.

The calcium-dependent oxygen evolution activity of preparations of Phormidium luridum shows a marked selectivity in favor of ferricyanide over benzoquinone as Hill oxidant. In addition, the rate of oxygen evolution increases with increasing solution redox potential over the range +350 to +550 mV vs. the standard hydrogen electrode. These properties pertain to both 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive and -insensitive fractions of the total oxygen evolution activity. Neither changes in solution potential nor use of oxidants other than ferricyanide obviate the need for added Ca(2+). To explain these observations, two models are proposed, each of which invokes the existence of a redox component located within Photosystem II and having a midpoint potential greater than +450 mV. In one model, the postulated species is a donor which competes with water for oxidizing equivalents generated by System II. In the other model, the 450 mV species is a high-potential primary acceptor of System II electrons.     

Na(+)-Ca2+ exchange activity in synaptic plasma membranes derived from the electric organ of Torpedo ocellata.

Synaptic plasma membranes obtained by hypo-osmotic treatment of purified Torpedo ocellata synaptosomes, contain an electrogenic Na(+)-Ca2+ exchange system. The dependence of the initial reaction rate on [Ca2+] reveals a single binding site for Ca2+ with an average apparent Km of 13.66 (S.D. = 12.07) microM [Ca2+] and maximal reaction velocity of Vmax = 11.33 (S.D. = 5.93) nmol/mg protein per s. The dependence of the initial rate of the Na+ gradient dependent Ca2+ influx on the internal [Na+] exhibits a sigmoidal curve which reaches half-maximal reaction rate at 170.8 (S.D. = 19.9) mM [Na+]. Addition of ATP gamma S does not change the K0.5 to Na+. The average Hill coefficient is 3.09 (S.D. = 0.86) indicating that 3-4 Na+ ions are exchanged for each Ca2+. Na+ gradient dependent Ca2+ uptake in Torpedo SPMs takes place also in the absence of K+ suggesting that K+ co-transport is not obligatory. The temperature dependence of the initial and steady-state rates of Na+ gradient dependent Ca2+ influx reveal that maximal reaction velocities of the Torpedo exchanger are attained between 15 and 20 degrees C. The energy of activation between 0 and 20 degrees C is 20,826 cal/mol. In comparison, rat brain synaptic plasma membrane Na(+)-Ca2+ exchanger reaches maximal reaction rates between 30 and 40 degrees C. Reconstitution of Torpedo or rat brain Na(+)-Ca2+ exchangers into a membrane composed of either Torpedo or brain phospholipids, does not alter the temperature dependence of the native Torpedo or rat brain Na(+)-Ca2+ exchangers; inspite of considerable differences in the composition of the fatty acyl chains that are esterified to brain and Torpedo phospholipid head groups and differences in membrane fluidity that were detected. An ATP-dependent Ca2+ pump, which is insensitive to FCCP, is also present in the same synaptic membrane.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Sodium/calcium exchange in smooth-muscle microsomal fractions.

The existence of Na+ -dependent Ca2+ transport was investigated in microsomal fractions from the longitudinal smooth muscle of the guinea-pig ileum and from the rat aorta, and its activity was compared with that of the plasmalemmal ATP-dependent Ca2+ pump previously identified in these preparations. The rate of Ca2+ release from plasmalemmal vesicles previously loaded with Ca2+ through the ATP-dependent Ca2+ pump was transiently faster in the presence of 150 mM-NaCl in the medium than in the presence of 150 mM-KCl or -LiCl or 300 mM-sucrose. Na+-loaded vesicles took up Ca2+ when an outwardly directed Na+ gradient was formed across the membrane. The Ca ionophore A23187 induced a rapid release of 85% of the sequestered Ca2+, whereas only 15% was displaced by La3+. Ca2+ accumulated by the Na+-induced Ca2+ transport was released by the addition of NaCl, but not KCl, to the medium. Ca2+ uptake in Na+-loaded vesicles was inhibited in the presence of increasing NaCl concentration in the medium. Half-maximum inhibition was observed with 28 mM-NaCl. Data fitted the Hill equation, with a Hill coefficient (h) of 1.9. Na+-induced Ca2+ uptake was a saturable function of Ca2+ concentration in the medium. Half-maximum activity was obtained with 18 microM-Ca2+ in intestinal-smooth-muscle microsomal fraction and with 50 microM-Ca2+ in aortic microsomal fraction. The results suggest that in these membrane preparations a transmembrane movement of Ca2+ can be driven by a Na+ gradient. However, the Na+-induced Ca2+ transport had a lower capacity, a lower affinity and a slower rate than the ATP-dependent Ca2+ pump.     

Probing the functional role of Ca2+ in the oxygen-evolving complex of photosystem II by metal ion inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.     

Calmodulin-induced activation of ATP-dependent Ca2+ transport in plasma membranes of the myometrium

Calmodulin activates the ATP-dependent transport of Ca2+. The V0 value for this reaction in the absence of calmodulin is 0.82, that in the presence of 10(-7) M calmodulin is 5 times as high, i. e. 4.5 nmol 45Ca2+/mg protein/min. The Vmax value in the absence of calmodulin is 2.07, that with the activator is 4.33 nmol 45Ca2+/mg protein/min. The corresponding Km values are 0.75 X 10(-6) M and 0.66 X 10(-7) M, respectively, i. e., the affinity of the Ca-pump for Ca2+ increases. The half-maximum Ca-binding activity of calmodulin measured with a help of the fluorescent probe, N-phenyl-1-naphthylamine (PNA), is observed at 5 X 10(-7) M Ca2+. Mg2+ (3 mM) decreases 10-fold the Ca-binding affinity. No significant effect of ATP on the Ca-binding properties of calmodulin was found; the Hill coefficient is suggestive of a positive cooperativity of this reaction. A comparison of dependences of the calmodulin-stimulated component of ATP-dependent transport of Ca2+ in myometrium plasma membranes and of the Ca-binding activity of calmodulin measured with a help of PNA suggests that the effect of calmodulin on the affinity of the Ca-pump for Ca2+ can also be realized when some (but not all) Ca-binding sites in the calmodulin molecule are saturated with Ca2+.     

Kinetic studies on the activation of human factor X. The role of metal ions on the reaction catalyzed by the venom coagulant protein of Viper russelli

The effect of Ca2+, Mg2+, and Mn2+ on the initial rate of activation of human Factor X by the venom coagulant protein of Vipera russelli has been investigated. Neither Mg2+ nor Mn2+ alone support the reaction. Ca2+ is an essential activator and exhibits cooperative kinetics. Both Mg2+ and Mn2+ enhance the reaction cooperatively when Ca2+ is present at suboptimal concentrations. Similarly, Ca2+ quenches the intrinsic fluorescence of human Factor X in a cooperative manner. While neither Mg2+ nor Mn2+ by themselves affect the fluorescence of human Factor X, they decrease the cooperativity of the Ca2+ binding to the protein as judged by Hill plots of the Ca2+ -induced fluoresence quenching. EPR measurements indicate that there are three high affinity Mn2+ binding sites on human Factor X which can also bind Ca2+. Positive cooperativity was not observed for Mn2+ binding. These data indicate that Ca2+ can cause a conformational change of the Factor X molecule which allows the activation reaction to proceed. We propose that Mn2+ does not support the activation of human Factor X because it cannot induce a necessary conformational change in the absence of Ca2+.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Calcium retards NH2OH inhibition of O2 evolution activity by stabilization of Mn2+ binding to photosystem II

Calcium is required for oxidation of water to molecular oxygen by photosystem II; the Ca2+ demand of the reaction increases upon removal of 23- and 17-kDa extrinsic polypeptides from detergent-derived preparations of the photosystem. Employing the manganese reductant NH2OH as a probe to examine the function of Ca2+ in photosystem II reveals that (1) Ca2+ slows the rate of NH2OH inhibition of O2 evolution activity, but only in photosystem II membranes depleted of extrinsic proteins, (2) other divalent cations (Sr2+, Cd2+) that compete for the Ca2+ site also slow NH2OH inhibition, (3) Ca2+ is noncompetitive with respect to NH2OH, (4) in order to slow inhibition, Ca2+ must be present prior to the initiation of NH2OH reduction of manganese, and (5) Ca2+ appears not to interfere with NH2OH reduction of manganese. We conclude that the ability of Ca2+ to slow the rate of NH2OH inhibition arises from the site in photosystem II where Ca2+ normally stimulates O2 evolution and that the mechanism of this phenomenon arises from the ability of Ca2+ or certain surrogate metals to stabilize the ligation environment of the manganese complex.     

Isolation of an oxygen-evolving Photosystem II preparation containing only one tightly bound calcium atom from a chlorophyll b-deficient mutant of rice

Oxygen-evolving Photosystem II (PS II) particles were prepared from the thylakoid membranes of a chlorophyll b-less rice mutant, which totally lacks light-harvesting chlorophyll a/b proteins, after solubilization with β-octylglucoside. The preparation was essentially free of Photosystem I as judged from its low-temperature fluorescence spectrum and polypeptide composition. The PS II particles contained all the major subunit polypeptides of the PS II reaction center core complexes and the three extrinsic proteins related to oxygen evolution. The relative abundances of the 33, 21 and 15 kDa proteins were 100, 64 and 20%, respectively, of the corresponding proteins in the mutant thylakoids. The chlorophyll-to-Q_A ratio was 53 and there was only one bound Ca²⁺ per Q_A. Thus, one of the two bound Ca²⁺ present in the oxygen-evolving PS II membrane preparations from wild-type rice (Shen J.-R., Satoh, K. and Katoh, S. (1988) Biochim. Biophys. Acta 933, 358–364) is missing. The mutant PS II particles were highly active in oxygen evolution in the absence of exogenously added Ca²⁺, although addition of 5 mM Ca²⁺ enhanced the activity by 30%. When the 21 and 15 kDa proteins were supplemented to the particles, the Ca²⁺-effect disappeared and the rate of oxygen evolution increased to a level exceeding 1000 μmol O₂ per mg chlorophyll per h. The results indicate that the number of Ca²⁺ needed to promote a high rate of oxygen evolution is one per PS II in higher plants.