Lanthanum and some other cation-induced changes in fluidity of synaptosomal membrane studied with nitroxide stearate spin labels

Summary Using nitroxide fatty acid spin labels, the effects of some cations such as La³⁺, Cd²⁺ and Hg²⁺ on synaptosomal membranes were studied by observing changes in their ESR spectra. The labels were incorporated almost instantaneously into synaptosomes isolated from rat brain cortex. ESR spectra of the spin-labeled synaptosomes were significantly broadened immediately upon adding La³⁺, Ce³⁺, Cd²⁺ or Hg²⁺ but hardly affected by Ca²⁺, Sr²⁺ and Ba²⁺. The magnitude of the change in the separation of the outer two peaks in ESR spectra (2T ) depends on the number (n) of methylene units between the polar head group and the spin-label (nitroxide) group; that is, it increases with decreasingn. Among these ions, the effect of La³⁺ was the greatest and appeared to be in parallel with the amount of La³⁺ bound with the synaptosomes. On the other hand, K⁺, Rb⁺ or Li⁺ causes hardly any significant changes.     

'Slow' K+-stimulated Ca2+ influx is mediated by Na+-Ca2+ exchange: a pharmacological study

K+-stimulated 45Ca2+ influx was measured in rat brain presynaptic nerve terminals that were predepolarized in a K+-rich solution for 15 s prior to addition of 45Ca2+. This 'slow' Ca2+ influx was compared to influx stimulated by Na+ removal, presumably mediated by Na+-Ca2+ exchange. The K+-stimulated Ca2+ influx in predepolarized synaptosomes, and the Na+-removal-dependent Ca2+ influx were both saturating functions of the external Ca2+ concentration; and both were half-saturated at 0.3 mM Ca2+. Both were reduced about 50% by 20 microM Hg2+, 20 microM Cu2+ or 0.45 mM Mn2+. Neither the K+-stimulated nor the Na+-removal-dependent Ca2+ influx was inhibited by 1 microM Cd2+, La3+ or Pb2+, treatments that almost completely inhibited K+-stimulated Ca2+ influx in synaptosomes that were not predepolarized. The relative permeabilities of K+-stimulated Ca2+, Sr2+ or Ba2+ influx in predepolarized synaptosomes (10:3:1) and the corresponding selectivity ratio for Na+-removal-dependent divalent cation uptake (10:2:1) were similar. These results strongly suggest that the K+-stimulated 'slow' Ca2+ influx in predepolarized synaptosomes and the Na+-removal-dependent Ca2+ influx are mediated by a common mechanism, the Na+-Ca2+ exchanger.     

Acridine spin labels as probes for nucleic acids.

Adridine spin labels, 4-[9-(6-chloro-2-methoxy)-acridylamino]- 2,2,6,6-tetramethyl-1-piperidinyloxy (I) and 4-(9-acridylamino)- 2,2,6,6-tetramethyl-1-piperidinyloxy (II), have been synthesized and their interaction with nucleic acids studied by means of electron spin resonance (ESR). The ESR spectra of labels I and II in the presence of calf thymus DNA were characteristic of highly immobilized nitroxide radicals with maximum hyperfine splittings (2T_ˌˌ) of 58.7 and 55.5 G, respectively. The melting temperature (T_m) of DNA, determined in the presence of labels I and II by the ESR technique, were closely similar to those obtained by spectrophotometric methods. The ESR spectrum of label I bound to calf liver RNA and yeast RNA indicated that the nitroxide group of this label was highly mobile. These results suggest that spin labels I and II are suitable noncovalent probes for nucleic acids.     

二价金属离子对嵌有线粒体H~+-ATP酶的脂酶体不同层次脂质流动性的影响

本文报道用荧光偏振及顺磁共振两种方法研究Mg~(2+)及其它二价金属离子对嵌有H~+-ATP酶的脂酶体不同层次脂质流动性的影响。 (1)顺磁标记探剂5-、12-、16-氮氧基硬脂酸测定结果表明Mg~(2+)和其它二价金属离子都能降低膜脂双分子层表层的流动性。降低流动性的顺序为Mg~(2+)=Ca~(2+)>Sr~(2+)>Cd~(2+)。较深层脂则无明显变化。 (2)荧光探剂7-、12-(9-蒽酰)硬脂酸及16-(9-蒽酰)棕榈酸的测定结果也表明Mg~(2+)和其它二价金属离子降低了膜脂表层的流动性,尤以Mn~(2+)、Ca~(2+)降低流动性最显著,流动性降低的顺序为;Mn~(2+) Ca>Sr~(2+) Mg~(2+) Cd~(2+)。除Mn~(2+)、Ca~(2+)还能影响膜脂深层的流动性外,其它与对照无明显差异。     

Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex.

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.     

Calcium-Activated ATPases in Presynaptic Nerve Endings

We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.     

Metal ion-induced conformational changes in Escherichia coli alkaline phosphatase.

Ultraviolet difference spectra are produced by the binding of divalent metal ions to metal-free alkaline phosphatase (EC 3.1.3.1). The interaction of the apoprotein with Zn2+, Mn2+, Co2+ and Cd2+, which induce the tight binding of one phosphate ion per dimer, give distinctly different ultraviolet spectra changes from Ni2+ and Hg2+ which do not induce phosphate binding. Spectrophotometric titrations at alkaline pH of various metallo-enzymes reveal a smaller number of ionizable tyrosines and a greater stability towards alkaline denaturation in the Zn2+- and Mn2+-enzymes than in the Ni2+-, Hg2+- and apoenzymes. The Zn2+- and Mn2+-enzymes have CD spectra in the region of the aromatic transitions that are different from the CD spectra of the Ni2+-, Hg2+- and apoenzymes. Modifications of arginines with 2,3-butanedione show that a smaller number of arginine residues are modified in the Zn2+-enzyme than in the Hg2+-enzyme. The presented data indicate that alkaline phosphatase from Escherichia coli must have a well-defined conformation in order to bind phosphate. Some metal ions (i.e. Zn2+, Co2+, Mn2+ and Cd2+), when interacting with the apoenzyme, alter the conformation of the protein molecule in such a way that it is able to interact with substrate molecules, while other metal ions (i.e. Ni2+ and Hg2+) are incapable of inducing the appropriate conformational change of the apoenzyme. These findings suggest an important structural function of the first two tightly bound metal ions in enzyme.     

Selectivity of the Ca binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations

K-stimulated (voltage-dependent) influx of 45Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain. Influx was terminated at 1 s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D. A., and Blaustein, M. P., 1980, J. Gen. Physiol., 76:709- 728). This influx was blocked by multivalent cations with half- inhibition constants (K1) that clustered in three distinct groups: (a) K1 greater than 1 mM (Mg2+, Sr2+, and Ba2+); (b) K1 = 30-100 microM (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Hg2+); (c) K1 less than 1 micro M (Cd2+, Y3+, La3+ and the trivalent lanthanides, and Pb2+). Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization. The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s; inhibition was similar after 1 s or 30 min of exposure to these ions. The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation. The relative efficacies of the lanthanides as fast channel blockers were compared; there was a decrease in inhibitory potency with decreasing ionic radius. A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations. The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them. Small cations are unable to bind effectively to both anions. The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel.     

重金属对水稻和小麦DNA甲基化水平的影响

和对照相比,0．025（或0.05)-0.1mmol/L的Cu^2 （或0．05）－1．0mmol/L的Cd^2 或Hg^2 导致水稻（或小麦）叶DNA中的5－甲基胞嘧啶百分含量大幅度上升;当Cu^2 浓度＞0.1mmol/L时,小麦和水稻叶DNA中5－甲基胞嘧啶的百分含量随Cu^2 浓度的增高略有下降,但仍高于对照。0.1-1.0mmol/L的Cu^2 ,Cd^2 和Hg^2 也导致小麦穗DNA为5－甲基胞嘧啶的百分含量随Cu^2 ,Cd^ 和Cd^2 能使小麦和水稻根系DNA中5－甲基胞嘧啶的百分含量显著高于对照,而0.1-1.0mmol/L的Hg^2 以及1．0mmol/L的Cu^2 和Cd^2 则造成小麦和水稻根系DNA中5－甲基胞嘧啶的百分含量显著低于对照。     

Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N3-SL-ATP

2-N3-SL-ATP [2-azido-2',3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl-pyrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotide-binding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spin-spin interactions between two radicals corresponding to a distance of about 15 A between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca2+-ATPase is a dimer with the nucleotide-binding sites in close proximity.     

Differential effects of monovalent, divalent and trivalent metal ions on rat brain hexokinase

The effects of monovalent (Li+, Cs+) divalent (Cu2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Hg2+, Pb2+, Mn2+, Fe2+, Co2+, Ni2+) and trivalent (Cr3+, Fe3+, Al3+) metals ions on hexokinase activity in rat brain cytosol were compared at 500 microM. The rank order of their potency as inhibitors of brain hexokinase was: Cr3+ (IC50 = 1.3 microM) greater than Hg2+ = Al3+ greater than Cu2+ greater than Pb2+ (IC50 = 80 microM) greater than Fe3+ (IC50 = 250 microM) greater than Cd2+ (IC50 = 540 microM) greater than Zn2+ (IC50 = 560 microM). However, at 500 microM Co2+ slightly stimulated brain hexokinase whereas the other metal ions were without effect. That inhibition of brain glucose metabolism may be an important mechanism in the neurotoxicity of metals is suggested.     

Effects of cadmium and mercury on Na(+)-K+, ATPase and uptake of 3H-dopamine in rat brain synaptosomes.

Effects in vivo of cadmium (Cd), mercury (Hg) and methylmercury (CH3Hg) on Na(+)-K+ ATPase and uptake of 3H-dopamine (DA) in rat brain synaptosomes were studied. These heavy metals significantly inhibited the Na(+)-K+ ATPase activity in a dose-dependent manner. Similarly, inhibition of DA uptake by synaptosomes was also observed in rats treated with these metals. Intraperitoneal route of metal administration was found to be more effective than per os treatment. Mercuric compounds compared to Cd elicited a higher inhibition of Na(+)-K+ ATPase and DA uptake in rat brain synaptosomes.     

Novel spin-labels for the study of lipid-protein interactions. Application to (Na+, K+)-ATPase membranes

The interactions of a series of spin-labeled fatty acids, in which the nitroxide ring is incorporated in different ways as an integral part of the hydrocarbon chain, with the (Na+,K+)-ATPase in membranes from Squalus acanthias, have been studied by electron spin resonance spectroscopy. The fatty acids are 2,4-, 2,5-, and 3,2-substituents of 2,2,5,5-tetramethylpyrrolidine-N-oxyl and belong to the class of minimal perturbation nitroxide probes. For all five fatty acid labels, a motionally restricted lipid component was observed in the ESR spectra of (Na+,K+)-ATPase membranes, in addition to the fluid component, which was found in the spectra of the extracted membrane lipids. The pH dependence of the motionally restricted spin-label population indicated a sensitivity in the selectivity of the lipid-protein interaction to the protonation state of the fatty acid. These results agree with those found previously for the conventional oxazolidine (doxyl) fatty acid and phospholipid spin-label derivatives [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] and indicate that the motion of the lipid chains is significantly hindered by interaction with the protein, irrespective of the nature of the spin-label group.     

Interactions Between α-Latrotoxin and Trivalent Cations in Rat Striatal Synaptosomal Preparations

The interactions between alpha-latrotoxin (alpha-LTx), a neurosecretagogue purified from the venom of the black widow spider, and the trivalent cations Al3+, Y3+, La3+, Gd3+, and Yb3+ were investigated in rat striatal synaptosomal preparations. All trivalent cations tested were inhibitors of alpha-LTx-induced [3H]dopamine [( 3H]DA) release (order of potency: Yb3+ greater than Gd3+ approximately Y3+ greater than La3+ greater than Al3+). Only with Al3+ could inhibition of [3H]DA release be attributed to a block of 125I-alpha-LTx specific binding to synaptosomal preparations. The inhibitory effect of trivalent ions was reversible provided synaptosomes were washed with buffer containing EDTA. Trivalent ions also inhibited alpha-LTx-induced [3H]DA release at times when alpha-LTx-stimulated release was already evident. alpha-LTx-induced synaptosomal membrane depolarization was blocked by La3+, but not affected by Gd3+, Y3+, and Yb3+. alpha-LTx-stimulated uptake of 45Ca2+ was inhibited by all trivalent cations tested. These results demonstrate that there exist at least three means by which trivalent cations can inhibit alpha-LTx action in rat striatal synaptosomal preparations: (1) inhibition of alpha-LTx binding (Al3+); (2) inhibition of alpha-LTx-induced depolarization (La3+); and (3) inhibition of alpha-LTx-induced 45Ca2+ uptake (Gd3+, Y3+, Yb3+, La3+).     

超氧化物歧化酶的自旋标记研究

本文应用对-SH基特异性结合的自旋标记物对Cu_2Zn_2-SOD进行自旋标记研究,进一步观察了电离辐射对-SH基部位的影响,实验结果:(1)自旋标记后的Cu_2Zn_2-SOD表现出典型的弱固定化的ESR波谱信号特征。标记对Cu_2Zn_2-SOD的UV谱无明显影响。这表明该游离-SH基是位于酶蛋白分子的相对表层而不是包埋在疏水内部。(2)标记保温后立即取样及保温后17小时取样测酶活力,发现与未标记酶的活力相同。这表明该-SH基与酶的催化活性无直接关系。(3)在10—100Krad γ-射线照射下,酶活性及ESR信号幅度均随照射剂量增加而下降,两者之间有一定的相关性。     

Bacterial sorption of heavy metals

Four bacteria, Bacillus cereus, B. subtilis, Escherichia coli, and Pseudomonas aeruginosa, were examined for the ability to remove Ag+, Cd2+, Cu2+, and La3+ from solution by batch equilibration methods. Cd and Cu sorption over the concentration range 0.001 to 1 mM was described by Freundlich isotherms. At 1 mM concentrations of both Cd2+ and Cu2+, P. aeruginosa and B. cereus were the most and least efficient at metal removal, respectively. Freundlich K constants indicated that E. coli was most efficient at Cd2+ removal and B. subtilis removed the most Cu2+. Removal of Ag+ from solution by bacteria was very efficient; an average of 89% of the total Ag+ was removed from the 1 mM solution, while only 12, 29, and 27% of the total Cd2+, Cu2+, and La3+, respectively, were sorbed from 1 mM solutions. Electron microscopy indicated that La3+ accumulated at the cell surface as needlelike, crystalline precipitates. Silver precipitated as discrete colloidal aggregates at the cell surface and occasionally in the cytoplasm. Neither Cd2+ nor Cu2+ provided enough electron scattering to identify the location of sorption. The affinity series for bacterial removal of these metals decreased in the order Ag greater than La greater than Cu greater than Cd. The results indicate that bacterial cells are capable of binding large quantities of different metals. Adsorption equations may be useful for describing bacterium-metal interactions with metals such as Cd and Cu; however, this approach may not be adequate when precipitation of metals occurs.     

Metal-ion binding to parvalbumin. A 113Cd-n.m.r. study of the binding of different lanthanide ions.

113Cd-n.m.r. studies were used to investigate the binding of the lanthanide ions La3+, Gd3+, Tb3+, Yb3+ and Lu3+ to parvalbumins. It was shown that lanthanide ions with a smaller ionic radius bind sequentially to Cd2+-saturated parvalbumin, whereas those with a larger ionic radius bind with similar affinity to both the CD site and the EF site. The smallest ion, Lu3+, does in fact not compete significantly with Cd2+ for the CD site in carp parvalbumin, but appears to bind only to the EF site. This preference of the smaller lanthanide ions for the EF site was used to assign the n.m.r. signals for protein-bound 113Cd. By using Cd n.m.r. and Tb3+ fluorescence it was also shown for alpha-lineage parvalbumin from pike that these proteins possess a third site that can bind lanthanide ions. This site is, however, much weaker than in the beta-lineage parvalbumins. It was used to assign the 113Cd resonances from protein-bound Cd2+ ions in the spectrum of pike pI5.0 parvalbumin.     

EPR studies show that all lanthanides do not have the same order of binding to calmodulin

Calmodulin, spin labeled at Tyr-99, has been titrated with the lanthanides La3+, Nd3+, Eu3+, Tb3+, Er3+ and Lu3+ as well as Ca2+ and Cd2+. The titration was monitored by EPR and changes in mobility of the spin label, due to binding into the labeled site and protein conformational change, were observed. Comparison of these titration curves with theoretical binding curves for the various calmodulin-metal species, show that different lanthanides have different high affinity sites. Three basic categories were observed, with Lu3+ and Er3+ behaving like Ca2+, Eu3+ and Tb3+ binding in the opposite order from Ca2+, and La3+ and Nd3+ different from either Ca2+ or Tb3+.     

Bacterial sorption of heavy metals.

Four bacteria, Bacillus cereus, B. subtilis, Escherichia coli, and Pseudomonas aeruginosa, were examined for the ability to remove Ag+, Cd2+, Cu2+, and La3+ from solution by batch equilibration methods. Cd and Cu sorption over the concentration range 0.001 to 1 mM was described by Freundlich isotherms. At 1 mM concentrations of both Cd2+ and Cu2+, P. aeruginosa and B. cereus were the most and least efficient at metal removal, respectively. Freundlich K constants indicated that E. coli was most efficient at Cd2+ removal and B. subtilis removed the most Cu2+. Removal of Ag+ from solution by bacteria was very efficient; an average of 89% of the total Ag+ was removed from the 1 mM solution, while only 12, 29, and 27% of the total Cd2+, Cu2+, and La3+, respectively, were sorbed from 1 mM solutions. Electron microscopy indicated that La3+ accumulated at the cell surface as needlelike, crystalline precipitates. Silver precipitated as discrete colloidal aggregates at the cell surface and occasionally in the cytoplasm. Neither Cd2+ nor Cu2+ provided enough electron scattering to identify the location of sorption. The affinity series for bacterial removal of these metals decreased in the order Ag greater than La greater than Cu greater than Cd. The results indicate that bacterial cells are capable of binding large quantities of different metals. Adsorption equations may be useful for describing bacterium-metal interactions with metals such as Cd and Cu; however, this approach may not be adequate when precipitation of metals occurs.     

Cytogenetical Toxical Effects of Heavy Metals on Vicia faba and Inquires into the Vicia Micro nucleus

Under the treatment of heavy metal ions Pb2+, Cd2+ and Hg2+, the interval of mitotic stage was shortened, and the time of interphase prolonged so that the cell cycle was prolonged in the root-tip cells of broadbean (Vicia faba L. ). With the increase of concentrations of Pb2+, Cd2+ and Zn2+ below 1.0, 0.01 and 10 ppm respectively, the mitosis index (IM) rose in root-tip cells, but IM decreased when the root-tips were treated with the some heavy metal ions above the above-mentioned respective concentrations. IM was inhibited in Hg2+ of any concentrations. Within the concentration of Pb2+, Cd2+, Hg2+ and Zn2+ below 1.0, 0. 50, 5.0, 100.0 ppm respectively, the frequency of micronucleus (MCNF) rose as the concentrations were increased, and lowered as the respective concentrations exceeded those stated above. Similar changes occurred in the frequency of chromosomal aberrations (CAF) when the concentration of Pb2+, Cd2+, Hg2+, Zn2+ were below or above 5.0, 5.0, 0.50, 100.0 ppm respectively. Mn2+ had no significant effects to them. By data processing with the method of gray system control and computer aided drawing to IM, MCNF and CAF, it was shown that the three parameters varied tremendously in different dose-effect ranges. All of which suggested that in order to obtain a reliable results in the environmental monitoring and hazardous material detection, genetoxicity inspection should be carried out under the optimal condition when (1) the concentration of the heavy metal to be detected does not seriously inhibit mitosis and (2) CAF and MCNF is in positive correlation.