43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.     

67Zn and 1H NMR studies of Zn2+-imidazole and carboxylate complexes

⁶⁷Zn NMR studies of naturally abundant Zn²⁺-imidazole and carboxylate ligands Complexes are shown. Thus, quadrupolar relaxation changes in ⁶⁷Zn NMR caused by adding imidazole ligands are more remarkable than those by carboxylate ligands. The changes caused by adding less bulky imidazole ligands are more prominent than those caused by a bulky imidazole ligan. Changes in Zn²⁺ quadrupolar relaxation rate caused by adding a cyclic hexapeptide consisting of L-histidine, L-cystein(Acm) and D-leucine are larger than those by a corresponding linear hexapeptide. Those changes in the quadrupolar relaxation rate of ⁶⁷Zn NMR among Zn²⁺ complexes can be reasonably interpreted in terms of the differences of equilibrium constants of those complexes to a first approximation.     

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.     

Zn2+, Mg2+, and H+ binding to d-fructose 1,6-bisphosphate studied by 31P and 1H NMR

The anomeric composition and mutarotation rates of fructose 1,6-bisphosphate were determined in the presence of 100 mm KCl at pH 7.0 by ³¹P NMR. At 23 and 37 °C the solution contains (15 ± 1)% of the α anomer. The anomeric rate constants at 37 °C are (4.2 ± 0.4) s^?1 for the β → α anomerization and (14.9 ± 0.5) s^?1 for the reverse reaction. A D₂O effect between 2.1 and 2.6 was found. From acid base titration curves it appeared that the pK values of the phosphate groups range from 5.8 to 6.0. Mg²⁺ and Zn²⁺ bind preferentially to the 1-phosphate in the α-anomeric position. Zn²⁺ has a higher affinity for this phosphate group than Mg²⁺ has. At increasing pH the fraction α anomer decreases slightly. At increasing Mg²⁺/fructose 1,6-bisphosphate ratios the fraction α anomer increases till 19% at a ratio of 20. Proton and probably Mg²⁺ binding decreases the anomerization rate. The time-averaged preferred orientation of the 1-phosphate along the C₁O₁ bond of the α conformer is strongly pH dependent, gauche rotamers being predominant at pH 9.4. In the presence of divalent cations the orientation is biased toward trans. A mechanistic model is proposed to explain the Zn²⁺, Mg²⁺, and pH-dependent behavior of the gluconeogenic enzyme fructose 1,6-bisphosphatase.     

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.     

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.     

25Mg NMR study of Mg2+-ATP,ADP-creatine kinase complexes

²⁵Mg NMR spectroscopy was first applied to the ternary complexes consisting of Mg²⁺, ATP, ADP and creatine kinase. The ²⁵Mg NMR spectra of the Mg²⁺-ATP (or ADP) complex are remarkably broadened in the ternary Mg²⁺-ATP(or ADP)-creatine kinase complex in contrast with previous prediction. From temperature dependence of the spectra of the protein-bound ion, it is suggested that Mg²⁺ of the protein-bound Mg²⁺-ATP(or ADP) complex is not in the fast exchange regime. The ²⁵Mg NMR signal of the transition state analogue complex is narrower and less temperature-dependent than those of the ternary complex, suggesting that Mg²⁺ in the transition state analogue complex is in a more symmetrical environment or exchanges slower than that of the ternary complex.     

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⁺.     

Relative roles of Ca2+, Sr2+, Ba2+ and Mg2+ in controlling lens permeability characteristics

The effects of barium, strontium and magnesium upon lens permeability characteristics were studied in the presence and absence of 2 mM calcium in the bathing medium. Permeability characteristics were determined by measuring lens potential, resistance and ⁴²K efflux rates. Barium and strontium at equimolar concentrations to calcium were able to substitute for calcium in controlling lens sodium permeability. Magnesium was ineffective in this respect.Small changes in resistance and ⁴²K efflux rates occurred when calcium was eliminated from bathing solution containing either 2 mM barium or strontium. These changes were interpreted to be the result of an increase in lens permeability to potassium. When 2 mM strontium was added to calcium-containing solution, there was no significant change in the electrical or flux parameters of the lens. However, the addition of 2 mM barium to calcium-containing solution resulted in a 54% increase in lens resistance and a 13 mV depolarization. These observations indicated a barium-induced decrease in lens permeability to potassium, and this was confirmed by an observed decrease in ⁴²K efflux rate constant under similar experimental conditions.The rapid time course of all the observed changes implies that they are the result of changes in the permeability characteristics of membranes lying close to the surface of the lens.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

川楝素对K+、Ca2+通道活动及细胞内Ca2+浓度的调控

川楝素是我国学者从驱蛔中药中分离、鉴定的一个三萜化合物，已证明具选择地影响神经递质释放，有效地对抗肉毒中毒，促进细胞分化、凋亡，抑制肿瘤增殖，抑制昆虫发育和取食，影响K⁺、Ca²⁺通道活动等多种生物效应. 综述了证明川楝素抑制多种K⁺通道，选择地易化L型Ca²⁺通道和进而升高胞内Ca⁺浓度的研究资料，并对川楝素产生这些生物效应的机制进行了讨论.     

A raman study of the interaction of Mg2+, Ca2+, and Ba2+ ions with an acidic model membrane

The interaction of dipalmitoylphosphatidylgly cerol DPPG) liposomes with divalent ions of magnesium, calcium and barium has been investigated with laser-Raman spectroscopy over the temperature range of 0–60°C. The effect of Ca²⁺ ions was also investigated as a function of concentration. At a Ca²⁺/DPPG molar ratio of 0.1, the number of trans carbon to carbon bonds in the hydrocarbon domain of the phospholipid and the lateral order of the hydrocarbon chains was increased both below and above the gel to liquid crystal transition. At higher Ca²⁺ concentrations the number of trans bonds and the lateral order is further increased over the entire temperature range studied, while the transition disappears. Magnesium and barium ions have a much smaller ordering effect on the side-chain packing of DPPG liposomes. At a molar ratio of 0.3, the gel to liquid crystal transition is still discernible for DPPG liposomes in the presence of Ba²⁺ ions, but not in the presence of Mg²⁺ ions.     

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.     

Laser Raman study of internally perfused muscle fibers effect of Mg2+, ATP and Ca2+

Raman spectra of an intact muscle fiber and of internally perfused fibers in capillary tubes have been obtained. The use of internal perfusion has insured a good control of the concentration of Ca²⁺, Mg²⁺ and ATP. The comparaison of the spectra obtained with the two types of fibers shows that the muscle structure is well preserved in capillary tubes. In addition, it appears that the sarcomere length has no significant effect on the Raman spectrum of muscle fibers. Our results on perfused fibers demonstrate that a fiber can be kept in the relaxed state for several hours, then displaying an intact fiber spectrum, when the concentration of ATP, Mg²⁺ and Ca²⁺ is maintained at 5, 2 and 0 mM, respectively. Therefore ATP and Mg²⁺ do not affect the Raman spectrum of muscle fibers. When one of these components is removed, or when Ca²⁺ is added, contraction occurs and causes major spectral changes. These results are interpreted as being due to strong electrostatic interactions between basic and acidic residues during contraction, and to a change of the α-helical content, or of the orientation, of some of the contractile proteins.     

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.     

Role of Ca2+ and Mg2+ in neutrophil hexose transport

The influence of extracellular Ca²⁺ and Mg²⁺ on the transport of 2-deoxy-[³H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[³H]glucose uptake stimulated by two chemotactic factors (C5a and $\text{N-}\text{formylmethionylleucylphenylalanine}$) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca²⁺ and Mg²⁺ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca²⁺, but not Mg²⁺, supported optimal enhanced hexose transport induced by stimuli.Activation of 2-deoxy-D-[³H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca²⁺ into the cell (as exemplified by the action of the two ionophores), such Ca²⁺ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca²⁺, may be involved in mediating the activation of hexose transport induced by the latter stimuli.     

匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+ 胁迫的生长响应与阈限浓度

采用砂培法,研究了匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+胁迫的生长响应及阈限浓度,结果表明:种子萌发率随着4种重金属浓度的增加而下降。对株高的影响是当重金属浓度小于100mg/L时会促进株高生长,高于100mg/L则产生抑制作用。Cu2+显著抑制根系生长,并随浓度的增加抑制效应愈加显著;在Cu2+浓度为600mg/L时匍茎翦股颖的根长比对照下降了93.75%。Cu2+、Zn2+、Pb2+浓度小于200mg/L时会促进地上生物量的增加,但高于200mg/L时,地上生物量会随着3种重金属的增加而减少。Cu2+、Zn2+浓度小于100mg/L或Cd2+、Pb2+浓度小于200mg/L会增加叶绿素的含量,高浓度会降低叶绿素的含量;Cd2+在浓度为600mg/L时显著降低叶绿素含量,与对照相比,下降了43.55%。匍茎翦股颖生长的综合效应分析表明,匍茎翦股颖对Cu2+胁迫最敏感,具有较低的阈限浓度,而Zn2+胁迫对匍茎翦股颖的生长影响最小,阈限浓度相对较高。