Bicarbonate is a native cofactor for assembly of the manganese cluster of the photosynthetic water oxidizing complex. Kinetics of reconstitution of O2 evolution by photoactivation

Assembly of the inorganic core (Mn(4)O(x)Ca(1)Cl(y)) of the water oxidizing enzyme of oxygenic photosynthesis generates O(2) evolution capacity via the photodriven binding and photooxidation of the free inorganic cofactors within the cofactor-depleted enzyme (apo-WOC-PSII) by a process called photoactivation. Using in vitro photoactivation of spinach PSII membranes, we identify a new lower affinity site for bicarbonate interaction in the WOC. Bicarbonate addition causes a 300% stimulation of the rate and a 50% increase in yield of photoassembled PSII centers when using Mn(2+) and Ca(2+) concentrations that are 10-50-fold larger range than previously examined. Maintenance of a fixed Mn(2+)/Ca(2+) ratio (1:500) produces the fastest rates and highest yields of photoactivation, which has implications for intracellular cofactor homeostasis. A two-step (biexponential) model is shown to accurately fit the assembly kinetics over a 200-fold range of Mn(2+) concentrations. The first step, the binding and photooxidation of Mn(2+) to Mn(3+), is specifically stimulated via formation of a ternary complex between Mn(2+), bicarbonate, and apo-WOC-PSII, having a proposed stoichiometry of [Mn(2+)(HCO(3)(-))]. This low-affinity bicarbonate complex is thermodynamically easier to oxidize than the aqua precursor, [Mn(2+)(OH(2))]. The photooxidized intermediate, [Mn(3+)(HCO(3)(-))], is longer lived and increases the photoactivation yield by suppressing irreversible photodamage to the cofactor-free apo-WOC-PSII (photoinhibition).Bicarbonate does not affect the second (rate-limiting) dark step of photoactivation, attributed to a protein conformational change. Together with the previously characterized high-affinity site, these results reveal that bicarbonate is a multifunctional "native" cofactor important for photoactivation and photoprotection of the WOC-PSII complex.     

Bicarbonate accelerates assembly of the inorganic core of the water-oxidizing complex in manganese-depleted photosystem II: a proposed biogeochemical role for atmospheric carbon dioxide in oxygenic photosynthesis

The proposed role for bicarbonate (HCO(3)(-)) as an intrinsic cofactor within the water-oxidizing complex (WOC) of photosystem II (PSII) [Klimov et al. (1997) Biochemistry 36, 16277-16281] was tested by investigation of its influence on the kinetics and yield of photoactivation, the light-induced assembly of the functional inorganic core (Mn(4)O(y)Ca(1)Cl(x)) starting from the cofactor-depleted apo-WOC-PSII center and free Mn(2+), Ca(2+), and Cl(-). Two binding sites for bicarbonate were found that stimulate photoactivation by accelerating the formation and suppressing the decay, respectively, of the first light-induced assembly intermediate, IM(1) [apo-WOC-Mn(OH)(2)(+)]. A high-affinity bicarbonate site (K(D) 相似文献   

Bicarbonate requirement for the water-oxidizing complex of photosystem II

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.     

Calcium controls the assembly of the photosynthetic water-oxidizing complex: a cadmium(II) inorganic mutant of the Mn4Ca core

Perturbation of the catalytic inorganic core (Mn4Ca1OxCly) of the photosystem II-water-oxidizing complex (PSII-WOC) isolated from spinach is examined by substitution of Ca2+ with cadmium(II) during core assembly. Cd2+ inhibits the yield of reconstitution of O2-evolution activity, called photoactivation, starting from the free inorganic cofactors and the cofactor-depleted apo-WOC-PSII complex. Ca2+ affinity increases following photooxidation of the first Mn2+ to Mn3+ bound to the 'high-affinity' site. Ca2+ binding occurs in the dark and is the slowest overall step of photoactivation (IM1-->IM1* step). Cd2+ competitively blocks the binding of Ca2+ to its functional site with 10- to 30-fold higher affinity, but does not influence the binding of Mn2+ to its high-affinity site. By contrast, even 10-fold higher concentrations of Cd2+ have no effect on O2-evolution activity in intact PSII-WOC. Paradoxically, Cd2+ both inhibits photoactivation yield, while accelerating the rate of photoassembly of active centres 10-fold relative to Ca2+. Cd2+ increases the kinetic stability of the photooxidized Mn3+ assembly intermediate(s) by twofold (mean lifetime for dark decay). The rate data provide evidence that Cd2+ binding following photooxidation of the first Mn3+, IM1-->IM1*, causes three outcomes: (i) a longer intermediate lifetime that slows IM1 decay to IM0 by charge recombination, (ii) 10-fold higher probability of attaining the degrees of freedom (either or both cofactor and protein d.f.) needed to bind and photooxidize the remaining 3 Mn2+ that form the functional cluster, and (iii) increased lability of Cd2+ following Mn4 cluster assembly results in (re)exchange of Cd2+ by Ca2+ which restores active O2-evolving centres. Prior EPR spectroscopic data provide evidence for an oxo-bridged assembly intermediate, Mn3+(mu-O2(-))Ca2+, for IM1*. We postulate an analogous inhibited intermediate with Cd2+ replacing Ca2+.     

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.     

The effect of di- and trivalent metal ions on the binding of [3H-Tyr1, D-Ala2, D-Leu5] enkephalin to opiate receptors from the rat brain

The influence of Ca2+, Mg2+, Mn2+, Sr2+, La3+, Nd3+, Sm3+, Eu3+, and Gd3+ ions on the binding of labeled, stable enkephalin analogue, [3H-Tyr1, D-Ala2, D-Leu5]enkephalin, to opiate receptors of the rat brain membrane preparations has been investigated. The formation of the complex can be described by a scheme involving at least two independent binding sites. The high affinity site does not discriminate the divalent and trivalent metal ions: all examined cations enhanced the enkephalin affinity for this site. The ligand binding to the low affinity site is potentiated only by Mn2+, Mg2+, and lathanoides. The maximal concentration of the binding sites of the above two types is not affected by the cations. The increase in the ionic strength of the solution entails a decrease in the affinity of the ligand for the high affinity binding site. It is shown that the effect of both di- and trivalent metal cations on the [3H-Tyr1, D-Ala2, D-Leu3] enkephalin binding is mediated through one cation attachment site on the respective enkephalin receptor.     

Characterization of calcium binding to brush-border membranes from rat duodenum.

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.     

Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [3H]GBR 12783: Attempts to Characterize the Na+ Dependence of the Neuronal Transport of Dopamine

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.     

Specific binding of tritium-labeled inositol 1,4,5-trisphosphate to human platelet membranes: ionic and GTP regulation.

Specific, saturable and reversible binding of tritium-labeled inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) to human platelet membranes is demonstrated. The Ins(1,4,5)P3-binding sites are abundant and display high selectivity for Ins(1,4,5)P3. Other inositol phosphates exhibit much lower affinity for this site. The specific [3H]Ins(1,4,5)P3 binding was found to be modulated by pH, monovalent and divalent cations, and GTP. A sharp increase in binding occurs at slightly alkaline pH. The monovalent cations, Na+, K+ and Li+ almost double the binding at 30 mM. Mg2+ inhibits the specific [3H]Ins(1,4,5)P3 binding. At low concentrations of Ca2+, the binding is inhibited, but at concentrations higher than 5 mM the binding is potentiated and increases by almost 5-fold at 100 mM. Similar pattern of the effects is also observed for Mn2+ and Sr2+. The specific [3H]Ins(1,4,5)P3 binding is specifically inhibited by GTP. Other nucleotides also inhibit the binding but at higher concentrations. From saturation binding studies, Ca2+ potentiation seems to be due to the conversion of the receptor from the low-affinity state to the high-affinity one. In the absence of Ca2+, the Scatchard plot is nonlinear and concave, and statistically can be fitted best with two equilibrium dissociation constants (Kd values), 0.19 +/- 0.11 and 13.2 +/- 18.1 nM, respectively, for high- and low-affinity binding sites. However, in the presence of 100 mM CaCl2, the Scatchard plot reveals only the high-affinity binding sites with a Kd value of 0.32 +/- 0.15 nM. The specific Ins(1,4,5)P3 receptor in human platelets could therefore exist in multiple conformational states to regulate the intracellular Ca2+ concentration.     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

Manganese and calcium requirements for reconstitution of oxygen-evolution activity in manganese-depleted photosystem II membranes

The Mn complex of photosystem II and O2-evolution activity are reconstituted in Mn-depleted photosystem II membranes in a light-dependent process called photoactivation. Recovery of O2-evolution activity requires both Mn2+ and Ca2+ in the photoactivation medium. The Mn2+ and Ca2+ dependences of both the effective rate constant and yield of photoactivation have been determined. A comparison of these data with the predictions of mathematical models for photoactivation leads to the conclusion that photoactivation occurs in two stages. The first stage, photoligation of Mn, requires light and depends primarily on Mn2+. The second stage, binding of Ca2+, is required for expression of O2-evolution activity. This two-stage model affords an excellent fit to the data and provides dissociation constants and binding stoichiometries for Ca2+ and Mn2+. We conclude that one Mn2+ ion is bound and photooxidized in the rate-determining step(s) of photoactivation. On the basis of these results and data already in the literature, the molecular details of the elementary steps in photoactivation are discussed and a mechanism of photoactivation is proposed.     

Spectroscopic evidence for Ca2+ involvement in the assembly of the Mn4Ca cluster in the photosynthetic water-oxidizing complex

Biogenesis and repair of the inorganic core (Mn4CaO(x)Cl(y)), in the water-oxidizing complex of photosystem II (WOC-PSII), occurs through the light-induced (re)assembly of its free elementary ions and the apo-WOC-PSII protein, a reaction known as photoactivation. Herein, we use electron paramagnetic resonance (EPR) spectroscopy to characterize changes in the ligand coordination environment of the first photoactivation intermediate, the photo-oxidized Mn3+ bound to apo-WOC-PSII. On the basis of the observed changes in electron Zeeman (g(eff)), 55Mn hyperfine (A(Z)) interaction, and the EPR transition probabilities, the photogenerated Mn3+ is shown to exist in two pH-dependent forms, differing in terms of strength and symmetry of their ligand fields. The transition from an EPR-invisible low-pH form to an EPR-active high-pH form occurs by deprotonation of an ionizable ligand bound to Mn3+, implicated to be a water molecule: [Mn3+ (OH2)] <--> [Mn3+ (OH-)]. In the absence of Ca2+, the EPR-active Mn3+ exhibits a strong pH dependence (pH approximately 6.5-9) of its ligand-field symmetry (rhombicity Delta delta = 10%, derived from g(eff)) and A(Z) (DeltaA(Z) = 22%), attributable to a protein conformational change. Binding of Ca2+ to its effector site eliminates this pH dependence and locks both g(eff) and A(Z) at values observed in the absence of Ca2+ at alkaline pH. Thus, Ca2+ directly controls the coordination environment and binds close to the high-affinity Mn3+, probably sharing a bridging ligand. This Ca2+ effect and the pH-induced changes are consistent with the ionization of the bridging water molecule, predicting that [Mn3+-(mu-O(-2))-Ca2+] or [Mn3+-(mu-OH(-))2-Ca2+] is the first light intermediate in the presence of Ca2+. The formation of this intermediate templates the apo-WOC-PSII for the subsequent rapid cooperative binding and photo-oxidation of three additional Mn2+ ions, forming the active water oxidase.     

Selective metal ion binding at the calcium-binding sites of the sea urchin extraembryonic coat protein hyalin

The interaction of metal ions with the sea urchin extraembryonic coat protein hyalin was investigated. Hyalin, immobilized on nitrocellulose membrane, bound Ca2+ and this interaction was disrupted by ruthenium red and selective metal ions. The divalent cations Cd2+ and Mn2+, when present at a concentration of 30 microM, displaced hyalin-bound Ca2+. In competition assays, 1 mM Cd2+ or 3 mM Mn2+ were effective competitors with Ca2+ for binding to hyalin. Cobalt, at a concentration of 30 microM, was unable to displace protein-bound Ca2+, but was effective in competition assays at a concentration of at least 10 mM. Magnesium and the monovalent cation Cs+ were unable to disrupt Ca2(+)-hyalin interaction. Interestingly, Cd2+, Mn2+, and Co2+ mimicked the biological effects of Ca2+ on the hyalin self-association reaction. These results clearly demonstrate that the Ca2(+)-binding sites on hyalin can selectively accommodate other divalent cations in a biologically active configuration.     

The effects of Mg2+ at the high-affinity and low-affinity sites on the polymerization of actin and associated ATP hydrolysis

Actin contains a single high-affinity cation-binding site, for which Ca2+ and Mg2+ can compete, and multiple low-affinity cation-binding sites, which can bind Ca2+, Mg2+, or K+. Binding of cations to the low-affinity sites causes polymerization of monomeric actin with either Ca2+ or Mg2+ at the high-affinity site. A rapid conformational change occurs upon binding of cations to the low-affinity sites (G----G) which is apparently associated with the initiation of polymerization. A much slower conformational change (G----G', or G----G' if the low-affinity sites are also occupied) follows the replacement of Ca2+ by Mg2+ at the high-affinity site. This slow conformational change is reflected in a 13% increase in the fluorescence of G-actin labeled with the fluorophore 7-chloro-4-nitrobenzene-2-oxadiazole (NBD-labeled actin). The rate of the ATP hydrolysis that accompanies elongation is slower with Ca-G-actin than with Mg-G'-actin (i.e. with Ca2+ rather than Mg2+ at the high-affinity site) although their rates of elongation are similar. The slow ATP hydrolysis on Ca-F-actin causes a lag in the increase in fluorescence associated with the elongation of actin labeled with the fluorophore N-pyrene iodoacetamide (pyrenyl-labeled actin), even though there is no lag in the elongation rate, because pyrenyl-labeled ATP-F-actin subunits have a lower fluorescence intensity than pyrenyl-labeled ADP-F-actin subunits. The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeled actin. Although their elongation rates are not very different, the rate of nucleation is much slower for Ca-G-actin than for Mg-G'-actin, probably because of the slower rate of ATP hydrolysis when Ca2+ is bound to the high-affinity site.     

Calcium-binding of Synaptosomes isolated from rat brain cortex. II. Inhibitory effects of magnesium ions and some other cations.

As in our previous report (Kamino, Uyesaka & Inouye, J. Membrane Biol. 17:13 1974), the absorbance changes of murexide caused by Ca2+ and followed up by a dual wavelength spectrophotometer were applied to measure synaptosomal Ca2+-binding in the presence of cations such as Rb+, Mn2+ or La3+. All the cations tested showed a significant inhibition of synaptosomal Ca2+-binding except Li+. The inhibitory effects could be divided into the following three categories: (1) noncompetive, co-operative K+-type, which includes alkali metal ions. The potency of inhibition is K+ greater than Rb+ greater than Cs+ greater than Li+, Na+ =0; (2) competitive Mn2+ -type which includes many divalent cations. The inhibitory potency was found to be in the following order: Mn2+ greater than Sr2+ greater than Cd2+, Ba2+ greater than Mg2+; (3) nonspecific, noncompetitive La3+ -type; among the cations tested, La3+ and Ce3+ were found to markedly reduce the Ca-binding capacity of synaptosomal particles, resulting in a noncompetitive inhibition, at least in the range of Ca2+ concentration used.     

Calorimetric experiments of Mn2+ -binding to alpha-lactalbumin

We measured by batch microcalorimetry the standard enthalpy change delta H degrees of the binding of Mn2+ to apo-bovine alpha-lactalbumin; delta H degrees = -90 +/- k J.mol-1. The binding constants, KMn2+, calculated from the calorimetric and circular dichroism titration curves, are (4.6 +/- 1).10(5) M-1 and (2.1 +/- 0.4).10(5) M-1, respectively. Batch calorimetry confirms the competitive binding Ca2+, Mn2+ and Na+ to the same site. The relatively small enthalpy change for Mn2+ binding compared to Ca2+ binding favours a model of a rigid and almost ideal Ca2+-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an alpha-helix which is directly connected to the complexating loop.     

Unique binding site for Mn2+ ion responsible for reducing an oxidized YZ tyrosine in manganese-depleted photosystem II membranes.

Binding of Mn2+ to manganese-depleted photosystem II and electron donation from the bound Mn2+ to an oxidized YZ tyrosine were studied under the same equilibrium conditions. Mn2+ associated with the depleted membranes in a nonsaturating manner when added alone, but only one Mn2+ ion per photosystem II (PS II) was bound to the membranes in the presence of other divalent cations including Ca2+ and Mg2+. Mn2+-dependent electron donation to photosystem II studied by monitoring the decay kinetics of chlorophyll fluorescence and the electron paramagnetic resonance (EPR) signal of an oxidized YZ tyrosine (YZ+) after a single-turnover flash indicated that the binding of only one Mn2+ ion to the manganese-depleted PS II is sufficient for the complete reduction of YZ+ induced by flash excitation. The results indicate that the manganese-depleted membranes have only one unique binding site, which has higher affinity and higher specificity for Mn2+ compared with Mg2+ and Ca2+, and that Mn2+ bound to this unique site can deliver an electron to YZ+ with high efficiency. The dissociation constant for Mn2+ of this site largely depended on pH, suggesting that a single amino acid residue with a pKa value around neutral pH is implicated in the binding of Mn2+. The results are discussed in relation to the photoactivation mechanism that forms the active manganese cluster.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

High-Affinity Cannabinoid Binding Site: Regulation by Ions, Ascorbic Acid, and Nucleotides

The high-affinity cannabinoid site in rat brain is an integral component of brain membranes that recognizes cannabinoids with inhibitory constants (Ki) in the nanomolar range. To clarify its physiological role, we studied the regulation of [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) binding. The site is inhibited by heavy metal ions, such as La3+, at low micromolar concentrations; divalent cations, such as Ca2+ and Mg2+, inhibit [3H]TMA binding, though at somewhat higher concentrations. In contrast, [3H]TMA binding is stimulated by Fe2+, Cu2+, and Hg2+ ions. Ascorbic acid and its analogs are also stimulators of cannabinoid binding at low micromolar concentrations. Stimulation of [3H]TMA binding by ascorbate or ions is dependent upon molecular oxygen, but is not inhibited by metabolic poisons. Metabolically stable nucleoside triphosphate analogs enhance [3H]TMA binding by different mechanisms, with hydrolysis of a high-energy phosphate bond apparently requisite for these influences. These results suggest that the cannabinoid binding site is associated with a nucleotide-utilizing protein possessing multiple regulatory subsites.