Synergetic effects of Ca2+ and Cu2+ on phase transition in phosphatidylserine membranes

In complexes of divalent metals with large exchange rate constant (K_H2O) of the coordinated H₂O, such as Ca²⁺ and Cu²⁺, the cubic structure in the ligand field is usually unstable and conformation changes are easily induced. We observed the molecular motion of phosphatidylserine (PS) in an amphipathic solvent (water / methanol / chloroform) by ¹H-NMR and ESR using Ca²⁺ and / or Cu²⁺, which has a similar K_H2O to that of Ca²⁺. We found that Ca²⁺ did not hinder the molecular movements of PS. However, Cu²⁺ reduced the movements of both headgroups and the double bonds in the fatty acids of PS. By addition of both Ca²⁺ and Cu²⁺, phase transition to a soft solid phase in the PS membrane was observed at room temperature. The results indicate that the headgroups are clustered in two-dimensional network with each ligand field displaced from the aqueous phase to the water / oil interface. The structure changes of the polar headgroups after the binding of divalent cations are considered to trigger the phase transition of this acidic phospholipid membrane.     

Interaction of Na+ and Mg2+ with Ca2+ in pancreatic islets as visualized by chlorotetracycline fluorescence

The fluorescence of microdissected pancreatic islets of ob/ob-mice was studied by microscope photometry after incubation with 10 μM chlorotetracycline. In Krebs-Ringer bicarbonate buffer, excitation at 390 nm yielded peak emission at 530 nm, suggesting that chelated Ca²⁺ was the major source of fluorescence. In support of this interpretation, incubation in Ca²⁺-free buffer markedly decreased the fluorescence, whereas withdrawal of Mg²⁺ increased it. Raising the Mg²⁺ concentration to 15 mM suppressed the fluorescence. In the presence of Ca²⁺, the substitution of choline ions for Na⁺ increased the fluorescence considerably; in the absence of Ca²⁺, however, Na⁺ deficiency had only little effect. Control experiments showed that Na⁺ or choline ions had no effect on the fluorescence of Ca²⁺-chlorotetracycline in 70 or 90% methanol. In 90%, but not in 70%, methanol 15 mM Mg²⁺ slightly quenched the fluorescence from 2.5 mM Ca²⁺ and 10 μM chlorotetracycline. It is suggested that Na⁺, and perhaps Mg²⁺, tends to decrease the amount of membrane-bound Ca²⁺ in the pancreatic islets.     

Transformation of (Ca2+ + Mg2+)ATPase of calmodulin-depleted erythrocyte membranes into a high Ca2+-affinity form by Ca2+

Specific activity and Ca²⁺-affinity of (Ca²⁺+Mg²⁺)ATPase of calmodulin-depleted ghosts progressively increase during preincubation with 0.1–2 mM Ca²⁺. Concomitantly, the increment in ATPase activity caused by calmodulin and the binding of calmodulin to ghosts decrease. The effects of calcium ions are abolished by the addition of calmodulin. ATP protects the enzyme from a Ca²⁺-dependent decrease of the maximum activity but does not seem to influence the Ca²⁺-dependent transformation of the low Ca²⁺-affinity enzyme into a high Ca²⁺-affinity form.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Synthetic peptide K+ carrier with Ca2+ inhibition

Based on a proposed solution conformation of the Ca²⁺ ion complex of the repeat hexapeptide of elastin, l-Val-l-Ala-l-Pro-Gly-l-Val-Gly, it is possible to modify the molecule making it more lipophilic for lipid bilayer permeation while retaining its complexation features. Therefore the two peptides, For-MeVal-Ala-Pro-Sar-Pro-Sar-OMe and For-MeVal-Ala-Pro-Sar-Pro-Sar-OH, were synthesized and evaluated for lipid bilayer activity and cation binding (For, N-formyl; Me, N-methyl; Sar, N-methyl glycine). Both peptides bound Ca²⁺ preferentially but did not exhibit the properties of a Ca²⁺ carrier. They were however active as K⁺ carriers although K⁺ ion titration curves showed a much lower affinity for K⁺ than for Ca²⁺. The addition of Ca²⁺ or Mg²⁺ to the bilayer system inhibited the peptide K⁺ carrier activity. Three possible explanations of this interesting Ca²⁺ inhibition of carrier activity are irreversible complexation of Ca²⁺, mixed ligand complex formation involving Ca²⁺, lipid and peptide, and impermeability of the lipid layer when peptide is complexed with a divalent cation.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Effect of calmodulin,Ca2+ and Mg2+ on the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes

Calmodulin-depleted isotonic erythrocyte ghosts contain 200 ng residual calmodulin/mg protein which is not removed by extensive washings at pCa²⁺ > 7. Specific activity and Ca²⁺-affinity of the (Ca²⁺ + Mg²⁺)ATPase increase at increasing calmodulin, with K_{0.5 Ca} of 0.38 μM at calmodulin concentrations corresponding to that in erythrocytes. High Ca²⁺ concentrations inhibit the enzyme. Specific activity and Ca²⁺-affinity of the enzyme decrease at increasing Mg²⁺ concentrations. The Ca²⁺ ? Mg²⁺ antagonism is likewise observed at inhibitory Ca²⁺ concentrations.     

Some effects of Ca2+, Mg2+, and Mn2+ on the ultrastructure,light-scattering properties,and malic enzyme activity of adrenal cortex mitochondria

The ultrastructure and 90 ° light-scattering capacity of adrenal cortex mitochondria have been examined under conditions which lead to an activation of malic enzyme activity in these mitochondria. After isolation, the mitochondria display an aggregate ultrastructure which does not resemble the vesicular (orthodox) form normally seen in vivo. Under conditions of malic enzyme activation (presence of malate, NADP⁺, Mg²⁺ and 1 mm Ca²⁺), the ultrastructure reverts to a vesicular form as seen in vivo. Of these required components, only Ca²⁺ affects the ultrastructure. The ultrastructural transformation from the aggregate to the orthodox form is always accompanied by a decrease in the 90 ° light-scattering capacity. When produced by Ca²⁺, transformation requires energy-dependent Ca²⁺ uptake if an oxidizable substrate is present. In the absence of substrate, the transformation occurs as an apparent energy-independent effect. Mn²⁺ can substitute for Ca²⁺ only in the presence of substrate. In de-energized mitochondria, Mn²⁺ prevents the effects of Ca²⁺. The activation of malic enzyme is always preceded by a decrease in light scattering and transformation to the orthodox ultrastructure; however, the presence of the orthodox form is not a sufficient condition since subsequent chelation of free Ca²⁺ fails to reverse either the decrease in light scattering or ultrastructural transformation but does reverse the enzyme activation. In addition, levels of Mn²⁺ which effectively depress light-scattering capacity and produce the orthodox form, fail to activate malic enzyme significantly. The data are discussed as they relate to Ca²⁺-induced damage in mitochondria.     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca²⁺泵(Ca²⁺-ATPase)是调节细胞内Ca²⁺浓度的重要蛋白质之一. Ca²⁺泵在转运Ca²⁺的过程中经历一系列构象变化. 其中,E1状态为外向的Ca²⁺高亲和状态,E2状态则为内向的Ca²⁺低亲和状态. 目前,骨骼肌内质网Ca²⁺泵转运Ca²⁺过程中的几个中间状态,包括E1-2Ca²⁺,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析. 介绍这几种状态的晶体结构,并分析Ca²⁺泵在执行功能过程中结构与功能的关系.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Comparative abilities of lanthanide ions La3+ and Tb3+ to substitute for Ca2+ in regulating phospholipid-sensitive Ca2+-dependent protein kinase and myosin light chain kinase

Although lanthanide ions La³⁺ and Tb³⁺ were only slightly able to substitute for Ca²⁺ to activate phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK), they potentiated the ability of a suboptimal concentration of Ca²⁺ to stimulate the enzyme. In comparison, the lanthanides were much more effective Ca²⁺ substitutes for myosin light chain kinase, a calmodulin-sensitive Ca²⁺-dependent protein kinase. Both enzymes, however, were inhibited by high concentrations of lanthanides either in the presence or absence of Ca²⁺. Similar effects of the lanthanides were also noted on phosphorylation of endogeneous substrates in the particulate fraction of rat brain stimulated by either phosphatidylserine/Ca²⁺ or calmodulin/Ca²⁺. The La³⁺- or Tb³⁺-stimulated activity of PL-Ca-PK, as the Ca²⁺-stimulated activity, was inhibited by various agents, such as trifluoperazine, polymyxin B, cobra cytotoxin I, melittin, and spermine.     

Effects of Cd2+, Mn2+, and Al3+ on rat brain synaptosomal uptake of noradrenaline and serotonin

Cd²⁺, Mn²⁺, and Al³⁺ inhibited synaptosomal amine uptake in a concentration-dependent and time-dependent manner. In the absence of Ca²⁺, the rank order of inhibition of noradrenaline uptake was: Cd²⁺ (IC₅₀ = 250 μM) > Al³⁺ (IC₅₀ = 430 μM) > Mn²⁺ (IC₅₀ = 1.50 mM), the IC₅₀ being the concentration of metal ions that gave rise to 50% inhibition of uptake. In the presence of 1 mM Ca²⁺, the rank order of inhibition of uptake was: Al³⁺ (IC₅₀ = 330 μM) > Cd²⁺ (IC₅₀ = 540 μM) > (IC₅₀ = 1.5 mM). The rank order of inhibition of serotonin uptake without Ca²⁺ was: Al³⁺ (IC₅₀ = 370 μM) > Cd²⁺ (IC₅₀ = 610 μM) > Mn²⁺ (IC₅₀ = 3.4 mM) and the rank order in the presence of 1 mM Ca²⁺ was: Al³⁺ (IC₅₀ = 290 μM) > Cd²⁺ (IC₅₀ = 1.5 mM) > Mn²⁺ (IC₅₀ = 4.0 mM). Ca²⁺, at 1 mM, definitely antagonized the inhibitory actions of Cd²⁺ on noradrenaline and serotonin uptake. Al³⁺ stimulated noradrenaline uptake at concentrations around 20–250 μM but inhibited this uptake at concentrations exceeding 300 μM in a dose-related fashion. Ca²⁺, at 1 mM, enhanced both the stimulatory and inhibitory effects of Al³⁺. Ca²⁺ also enhanced the inhibitory actions of Al³⁺ on seotonin uptake. These results, in conjunction with those we have previously published, suggest that Cd²⁺, Mn²⁺, and Al³⁺ exert differential and selective effects on the structure and function of synaptosomal membranes.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Na+-Ca2+ exchange activity in rabbit lymphocyte plasma membranes

Plasma membranes of rabbit thymus lymphocytes accumulated Ca²⁺ when a Na⁺ gradient (intravesicular > extravesicular) was formed across the membranes. Dissipation of the Na⁺ gradient by the addition of Na⁺ to the external medium decreased Ca²⁺ uptake. Ca²⁺ preloaded into the lymphocytes was extruded when Na⁺ was added to the external medium. The Ca²⁺ uptake decreased at acidic pH but increased at alkaline pH (above 8) and the activity was saturable for Ca²⁺ (apparent K_m for Ca²⁺ was 61 μM and apparent V_max was 11.5 nmol/mg protein per min). Na⁺-dependent uptake of Ca²⁺ was inhibited by tetracaine and verapamil, and partially inhibited by La³⁺. The uptake was not influenced by orthovanadate.     

Ca2+ and Mn2+ mediated binding of the glycoprotein peroxidase to membranes of Pharbitis cotyledons

Ca²⁺ and Mn²⁺ promote the binding of the basic isoperoxidase to a crude membrane preparation in extracts from Pharbitis cotyledons. The Ca²⁺- or Mn²⁺-induced binding is resistant to high ionic strength and can be saturated by increasing the divalent ion or the isoperoxidase concentrations. Treatments in vitro with glucosaminidase or in vivo with tunicamycin show that the carbohydrate part of the isoperoxidase is necessary for the binding. The amino sugar galactosamine inhibits the binding at rather high concentrations. Pharbitis basic isoperoxidase can be bound to zucchini squash microsomes in the presence of Ca²⁺ and conversely.     

Quantum chemical investigations on the complexes of Ca2+ and Zn2+ with aliphatic dipeptides

Several 1:1 complexes of Zn²⁺ with glycylglycine and of Ca²⁺ and Zn²⁺ with prolylglycine and glycylproline have been calculated within the Hartree-Fock method using a minimal GLO basis set. It was found that in spite of the fact that there are large differences in complex binding energy the relative stabilities of the different binding sites are the same in the case of Ca²⁺ and Zn²⁺ ions. Electronic density diagrams have been produced to illustrate the changes in electronic distribution caused by complex formation.