Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

Flash-induced consumption of molecular oxygen on the donor side of photosystem II in Mn-depleted subchloroplast membrane fragments: specific effects of manganese and calcium ions

It has been shown that removal of manganese from the water-oxidizing complex (WOC) of photosystem II (PSII) leads to flash-induced oxygen consumption (FIOC) which is activated by low concentration of Mn²⁺ (Yanykin et al., Biochim Biophys Acta 1797:516–523, 2010). In the present work, we examined the effect of transition and non-transition divalent metal ions on FIOC in Mn-depleted PSII (apo-WOC-PSII) preparations. It was shown that only Mn²⁺ ions are able to activate FIOC while other transition metal ions (Fe²⁺, V²⁺ and Cr²⁺) capable of electron donation to the apo-WOC-PSII suppressed the photoconsumption of O₂. Co²⁺ ions with a high redox potential (E ⁰ for Co²⁺/Co³⁺ is 1.8 V) showed no effect. Non-transition metal ions Ca²⁺ by Mg²⁺ did not stimulate FIOC. However, Ca²⁺ (in contrast to Mg²⁺) showed an additional activation effect in the presence of exogenic Mn²⁺. The Ca²⁺ effect depended on the concentration of both Mn²⁺ and Ca²⁺. The Ca effect was only observed when: (1) the activation of FIOC induced by Mn²⁺ did not reach its maximum, (2) the concentration of Ca²⁺ did not exceed 40 μM; at higher concentrations Ca²⁺ inhibited the Mn²⁺-activated O₂ photoconsumption. Replacement of Ca²⁺ by Mg²⁺ led to a suppression of Mn²⁺-activated O₂ photoconsumption; while, addition of Ca²⁺ resulted in elimination of the Mg²⁺ inhibitory effect and activation of FIOC. Thus, only Mn²⁺ and Ca²⁺ (which are constituents of the WOC) have specific effects of activation of FIOC in apo-WOC-PSII preparations. Possible reactions involving Mn²⁺ and Ca²⁺ which could lead to the activation of FIOC in the apo-WOC-PSII are discussed.     

Kinetic evidence for a dual cation role for muscle pyruvate kinase

The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg²⁺, Co²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg²⁺ and varying concentrations of Mn²⁺ or of Co²⁺. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co²⁺ and varying Mn²⁺. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn²⁺ is a better activator of the enzyme than is Mg²⁺, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni²⁺ or Zn²⁺ with Mg²⁺ suggests that Ni²⁺ and Zn²⁺ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg²⁺. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity.     

Toxicity of chromium and tin toAnabaena doliolum Interaction with bivalent cations

Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity ofAnabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca²⁺, Mg²⁺ and Mn²⁺, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca²⁺ > Mg²⁺ > Mn²⁺. However, the sequence of hierarchy was Mg²⁺ > Ca²⁺ > Mn²⁺ for carbon fixation and Mn²⁺ > Mg²⁺ > Ca²⁺ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth,¹⁴CO₂ uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity ofA. doliolum when Ni²⁺, Co²⁺ and Zn²⁺ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni²⁺ > Co²⁺ > Zn²⁺. Among all the interacting cations, Ca²⁺, Mg²⁺ and Mn²⁺ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni²⁺, CO²⁺ and Zn²⁺ showed synergistic inhibition which potentiating the toxicity of test metals in the N₂-fixing cyanobacteriumA. doliolum. It is evident from the present study that bivalent cations, viz. Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Zn²⁺, may appreciably regulate the toxicity of heavy metals in N₂-fixing cyanobacteria if present in aquatic media.     

Dual Control by Divalent Cations and Mitogenic Cytokines of α4β1 and α5β1 Integrin Avidity Expressed by Human Hemopoietic Cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function α4β1 and α5β1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by “inside-out” mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn²⁺, Mg²⁺ and Ca²⁺ for α4β1 and α5β1 activation by “inside-out” mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-β₁ integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of β₁ integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, α4β1 and α5β1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca²⁺, Mg²⁺ or Mn²⁺ were able to restore adhesive functions of α4β1 and α5β1 when activated by 8A2, only Mg²⁺ and Mn²⁺ were able to support activation of α5β1 and α5β1 by cytokines. Furthermore, high concentrations of Ca²⁺ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn²⁺ and cytokines but not by 8A2. On the contrary, in the presence of both Ca²⁺ and Mg²⁺, Mn²⁺ had an additive effect on the activation of α5β1 and α5β1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca²⁺, Mg²⁺ and Mn²⁺ may modulate in vivo α4β1 and α5β1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

Requirement of divalent cations for photoactivation of the laten water-oxidation system in intact chloroplasts from flashed leaves

The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn²⁺ (10 μM). (b) A high concentration of Mn²⁺ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca²⁺ (5 mM). (c) The Ca²⁺-dependent restoration was inhibited by a high concentration of Mg²⁺ or Sr²⁺, but the inhibition was restored by the coexistence of Ca²⁺. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca²⁺ inhibits photoactivation in competition with Mn²⁺. (ii) High concentration of Mn²⁺ inhibits photoactivation in competition with Ca²⁺. (iii) High concentration of Mg²⁺ affects photoactivation by inhibiting Ca²⁺-dependent restoration in competition with Ca²⁺. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn²⁺ and Ca²⁺, and that photoactivation takes place in the center having both Mn²⁺ and Ca²⁺ on their respective binding sites.     

Some properties of pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase

Reactivation of the pea mitochondrial pyruvate dehydrogenase complex was the result of dephosphorylation catalyzed by phospho-pyruvate dehydrogenase-phosphatase, an intrinsic component of the complex. Phosphatase activity was dependent upon divalent metal ions, with Mg²⁺ more effective than Mn²⁺ or Co²⁺. The Michaelis constants for Mg²⁺, Mn²⁺, and Co²⁺ were 3.8, 1.7, and 1.4 millimolar, respectively. Neither the rate nor the extent of activation of the phosphatase by Mg²⁺ or Mn²⁺ was effected by up to 100 units per assay of megamodulin. Calcium ions did not activate pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase, and low concentrations of Ca²⁺ antagonized activation by other divalent cations. Phosphatase activity was inhibited by fluoride and ortho-phosphate but not by molybdate or vanadate. Krebs cycle intermediates, adenylates, polyamines, amino acids, and phosphoamino acids were without effect upon pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase activity in vitro.     

Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Characterization of Plasma Membrane-associated Adenosine Triphosphase Activity of Oat Roots

ATPase activity of plasma membranes isolated from oat (Avena sativa L. cv. Goodfield) roots was activated by divalent cations (Mg²⁺ = Mn²⁺ > Zn²⁺ > Fe²⁺ > Ca²⁺) and further stimulated by KCl and a variety of monovalent salts, both inorganic and organic. The enzyme exhibited greater specificity for cations than anions. The presence of Mg²⁺ was necessary for KCl stimulation. Ca²⁺ was ineffective in replacing Mg²⁺ for activation of plasma membrane ATPase, but it did activate other membrane-bound ATPases. The pH optima for Mg²⁺ activation and KCl stimulation of the plasma membrane ATPase were 7.5 and 6.5, respectively.     

Mg2+ and Mn2+ modulation of Ca2+ transport and ATPase activity in sarcoplasmic reticulum vesicles

Kinetic experimentation was used to characterize the Mg²⁺ and Mn²⁺ modulation of Ca²⁺ transport and ATPase activity in sarcoplasmic reticulum vesicles. In addition to its participation in the ATP·Mg complex as substrate for the ATPase, Mg²⁺ is an activator of phosphoenzyme progression to hydrolylic cleavage. It is shown that this activation is due to Mg²⁺ occupancy of an allosteric site easily accessible on the outer surface of the vesicles, rather than to participation in an antiport mechanism. The Mg²⁺ site is distinct from the Ca²⁺ binding sites which are involved in activation of enzyme phosphorylation by ATP, and Ca²⁺ translocation. The role of Mg²⁺ is quite specific, inasmuch as phosphoenzyme decay is much slower if the Mg²⁺ allosteric site is occupied by Ca²⁺. Conversely, competive occupancy of the Ca²⁺ sites by Mg²⁺ does not permit enzyme phosphorylation by ATP. Intermediate characteristics between Mg²⁺ and Ca²⁺ are displayed by Mn²⁺ which is well able to stimulate phosphoenzyme cleavage by occupancy of the Mg²⁺ allosteric site, and is also able (although at much slower rates) to activate enzyme phosphorylation, and undergo active transport by occupancy of the Ca²⁺ sites.     

Evidence for the involvement of calmodulin in the operation of Ca-activated K channels in mouse fibroblasts

Summary The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca²⁺-activated K⁺ channels due to the oscillatory increase in the intracellular Ca²⁺ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 m, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca²⁺, Sr²⁺, Mn²⁺ and Ni²⁺, elicited slow hyperpolarizations, whereas Mg²⁺ and Ba²⁺, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca²⁺-activated K⁺ channels, abolished both Ca²⁺-and calmodulin-induced hyperpolarizations. TFP prevented Ca²⁺-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca²⁺-activated K⁺ channels in fibroblasts.     

Azospirillum brasilense glutamine synthetase: influence of the activating metal ions on the enzyme properties

The kinetic properties of the Mg²⁺-activated and Mn²⁺-activated glutamine synthetase (GS) of Azospirillum brasilense in the biosynthetic reaction were studied. The Mg²⁺-supported and Mn²⁺-supported GSs in an average state of adenylylation varied in pH optimum, maximum activity, saturation functions for ammonium and glutamate, affinity to substrates, and in the Me²⁺-ATP ratio required for the optimal enzyme activity. Seventeen other cations were tested for the maintenance of GS activity. The level of the latter and the kinetic behavior of the GS in A.brasilense is suggested to depend essentially on the concentrations of Mg²⁺, Mn²⁺ and Co²⁺, as well as on their ratio     

Comparison of the properties of the protein phosphatases from avian and mammalian smooth muscles: purification and characterization of rabbit uterine smooth muscle phosphatases

Three protein phosphatases were purified to near homogeneity from rabbit uterine muscle. These enzymes are termed rabbit uterine smooth muscle phosphatase (RU SMP)-I, -II, and -IV. RU SMP-I is composed of three subunits (Mr 60,000, 55,000, and 38,000) which comigrated with the subunits of turkey gizzard smooth muscle phosphatase (TG SMP)-I. Ethanol treatment of RU SMP-I dissociated the subunits and led to the purification of its catalytic subunit (Mr 38,000), RU SMP-Ic. Structural homology between the turkey gizzard and rabbit uterine SMP-I is indicated by the cross-reactivity of RU SMP-I with the polyclonal antibodies against TG SMP-I and -Ic. Like TG SMP-II, RU SMP-II is inactive in the absence of divalent cations and can be activated by Mg2+ and Mn2+. However, their electrophoretic profiles on sodium dodecyl sulfate-polyacrylamide gel are different. RU SMP-II shows two bands (Mr 42,000 and 44,000) while TG SMP-II is monomeric (Mr 43,000). Western blot analysis revealed that the 42,000 and 44,000-Da proteins cross-react with anti-TG SMP-II antibodies, suggesting that these proteins share common structural properties. The anti-TG SMP-I and Ic antibodies do not cross-react with RU SMP-II and -IV. Likewise, the anti-TG SMP-II antibodies do not cross-react with RU SMP-I and -IV, implying that these enzymes are distinct. RU SMP-IV is composed of a catalytic subunit (Mr 40,000) and a subunit with a molecular weight of 60,000 or 58,000. All three rabbit uterine smooth muscle phosphatases dephosphorylate the isolated myosin light chains but only RU SMP-IV dephosphorylates heavy meromyosin. However, when the catalytic subunit of RU SMP-I is dissociated from the regulatory subunits, it is active toward heavy meromyosin and exhibits higher activity toward myosin light chains and phosphorylase a than its holoenzyme. The substrate specificity of these enzymes and the effects of ATP, NaF, pyrophosphate, okadaic acid, Mg2+, Mn2+, and Ca2+ on their activities are very similar to those of the turkey gizzard smooth muscle phosphatases.     

The calculation of partial specific volumes of proteins in guanidine hydrochloride

The effects of various modifiers on the ATPase activity of bovine platelet actomyosin has been studied. The order of activation by monovalent cations was NH₄⁺? K⁺ > Li⁺ > Na⁺. The order of activation by divalent cations was Ca²⁺ > Mn²⁺ = Sr²⁺ > Ba²⁺> Co²⁺ > Mg²⁺ > Zn²⁺. Ethylenediaminetetraacetate inhibits. Activity increased with increasing concentrations of monovalent cations, except for inhibition by increasing concentrations of NH₄⁺ in the presence of Ca²⁺. Adenosine triphosphatase activity was increased by low concentrations of urea and trypsin, but was unaffected by low concentrations of N-ethylmaleimide. For all enzymatic properties where direct comparisons are possible, actomyosin from platelets is unlike that from skeletal muscle, but is similar to that from smooth muscle and non-muscle sources.     

Regulation of protein synthesis in rabbit reticulocyte lysates: effect of of Ca2+, Mg2+, and Mn2+ on self-phosphorylation and heterophosphorylation catalyzed by the heme-regulated and double-stranded-RNA-activated protein kinases

The influence of divalent cations Mg²⁺, Mn²⁺, and Ca²⁺ on the cyclic-AMP-independent protein kinases of the heine-regulated and double-stranded-RNA-activated translational inhibitory protein kinases on self-phosphorylation and heterophosphorylation of the substrate (the 38 000-dalton subunit of initiation factor eIF-2) has been examined, Results show that Mg²⁺, Mn²⁺, and Ca²⁺ affect the activities of these enzymes in the following fashion. Mg²⁺ supports both self-phosphorylation and heterophosphorylation efficiently. Mn²⁺ on the other hand supports self-phosphorylation but to a lesser degree the heterophosphorylation, Ca²⁺ promotes neither self-phosphorylation nor hetero-phosphorylation.     

Studies on the luminescent response of the Ca2+-activated photoprotein,obelin

1. The kinetics and stoicheiometry of the Ca²⁺-activated luminescent reaction of the photoprotein obelin were studied at different temperatures and in the presence of various substances, including the physiologically occurring cations K⁺, Na⁺, Ca²⁺, Mg²⁺ and H⁺. 2. The results suggest Ca²⁺-independent rates of rise and fall in obelin luminescence following sudden changes in [Ca²⁺] and indicate that changes in [Ca²⁺] over the range 1 · 10^?6?3 · 10^?4 M are followed significantly faster by the obelin response (approx. 3 ms delay at 20°C) than by the aequorin response (approx. 10 ms delay at 20°C). 3. Obelin was found to emit low-intensity light (less than 10^?6 of the maximum Ca²⁺-activated response), which was independent of Ca²⁺ at concentrations below about 10^?7 M. The level of this Ca²⁺-independent light emission is sensitive to temperature and the ionic composition of the solution. 4. The log-log plot of light intensity against ionized Ca indicates a maximum slope of 2.5, suggesting the involvement of three Ca ions in the luminescent reaction. 5. Increases in the concentration of K⁺, Na⁺, Mg²⁺ and H⁺ generally shift the Ca²⁺ activation curve for obelin toward higher Ca²⁺ concentrations. These cations can also affect the maximum rate of obelin utilization at more extreme concentrations. 6. The maximal rate of obelin utilization was also affected to varying degrees by the presence of uncharged substances such as glucose, sucrose and polyvinylpyrrolidone. However, neither the sensitivity of obelin to Ca²⁺ nor the quantum yield were modified by these substances. 7. Caffeine (less than 20 mM), procaine (less than 20 mM) and sodium dantrolene (saturated solution), substances known to modify cellular Ca²⁺ movements, had little effect on the Ca²⁺-induced luminescent reaction. The general anaesthetics chlorpromazine and halothane appeared to lower greatly the quantum yield without, however, modifying the maximum rate of obelin utilization. 8. A scheme of reaction for obelin activation by Ca²⁺ is presented which adequately explains the experimental observations and allows one to make accurate predictions regarding the relative obelin respones under a variety of ionic conditions at room temperature.     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Manganese enhances the phosphorylation of membrane-associated proteins isolated from barley roots

Microsomal membranes isolated from barley roots (Hordeum vulgare L. cv. CM72) contained endogenous protein phosphorylation activities that were greatly enhanced by Mn²⁺. Mg²⁺ions also stimulated protein phosphorylation, but to a lesser extent than Mn²⁺. Ca²⁺ enhanced Mg²⁺, but not Mn²⁺-dependent phosphorylation. It is proposed that this strong enhancement by Mn²⁺ may be due to a greater affinity of Mn²⁺ than either Ca²⁺ or Mg²⁺ for both the Ca²⁺ and Mg²⁺ binding sites of certain kinases. Some Mn²⁺ stimulated kinase activity was eliminated from the membrane by washing with 0.2 mol/L KCl. The KCl extract contained histone and casein kinase activities, and 4 major phosphoproteins that were phosphorylated on serine and threonine residues. Phosphorylation of a 52 kDa polypeptide corresponded with the characteristics of the histone kinase activity and may represent the autophosphorylation of a CDPK-type kinase. Phosphorylation of a 36 kDa polypeptide was Ca²⁺ stimulated and may represent the autophosphorylation of a different type of unknown kinase. Polypeptides of 18 and 15 kDa had characteristics that suggest they were autophosphorylating subunits of a membrane bound nucleotide di-phosphokinase.