Prevention by Ce3+ of DNA destruction caused by Hg2+ in fish intestine

The interactions between Hg²⁺, Ce³⁺, and the mixuure of Ce³⁺ and Hg²⁺, and DNA from fish intestine in vitro were investigated by using absorption spectrum and fluorescence emission spectrum. The ultraviolet absorption spectra indicated that the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ to DNA generated an obviously hypochromic effect. Meanwhile, the peak of DNA at 205.2 nm blue-shifted and at 258.2 nm red-shifted. The size of the hypochromic effect and the peak shift of DNA by metal ion treatments was Hg²⁺>Hg²⁺+Ce³⁺>Ce³⁺. The fluorescence emission spectra showed that with the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ the emission peak at about 416.2 nm of DNA did not obviously change, but the intensity reduced gradually and the sequence was Hg²⁺>Hg²⁺+Ce²⁺>Ce³⁺. Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ had 1.12, 0.19, and 0.41 binding sites to DNA, respectively; the fluorescence quenching of DNA caused by the metal ions all attributed to static quenching. The binding constants (K _A ) of binding siees were 8.98×10⁴ L/mol and 1.02×10⁴ L/mol, 5.12×10⁴ L/mol and 1.10×10³ L/mol, 6.66×10⁴ L/mol and 2.36×10³ L/mol, respectively. The results showed that Ce³⁺ could relieve the destruction of Hg²⁺ on the DNA structure.     

Combined actions of Pb2+, Zn2+, and Al3+ on voltage-activated calcium channel currents

Summary 1. Whole-cell patch clamp experiments were performed on rat dorsal root ganglion (DRG) neurons to investigate the actions of various combinations of Pb²⁺, Zn²⁺, and Al³⁺ on voltage-activated calcium channel currents (VACCCs).2. Each of these metals has been shown to reduce VACCCs.3. We investigated the effects of simultaneous application of two cations in the range of their IC₅₀ values. For all possible combinations (Pb²⁺/Zu²⁺, Zn²⁺/Al³⁺, Al³⁺/Pb²⁺), independent of the order of application, we found additive actions on VACCCs.4. We observed a 75% (±9%) block of the control current when two cations were applied simultaneously. This observation is consistent with both, an action of two metals at the same site as well as independent actions at different locations of the ion channel.5. The additivity of the effects should be taken into account for questions of public health and the assessment of threshold limits in cases of environmental contamination.     

Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K⁺ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K⁺ channel blocker TEA⁺ at a high concentration (20 mm). Barium, another classical K⁺ channel blocker, inhibited K⁺ currents with a K _i of about 1.3 mm. In contrast to guard cells, the cytosolic Ca²⁺ level proved to be ineffective in regulating the K⁺ conductance at hyperpolarization. External Ca²⁺ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating.A comparison between the K⁺ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca²⁺ to control K⁺ influx in xylem parenchyma cells is interpreted in physiological terms.     

Hg2+ and Cd2+ interact differently with biomimetic erythrocyte membranes

In order to characterize the potentially deleterious effects of toxic Hg²⁺ and Cd²⁺ on lipid membranes, we have studied their binding to liposomes whose composition mimicked erythrocyte membranes. Fluorescence spectroscopy utilizing the concentration dependent quenching of Phen Green™ SK by Hg²⁺ and Cd²⁺ was found to be a sensitive tool to probe these interactions at metal concentrations ≤1 μM. We have systematically developed a metal binding affinity assay to screen for the interactions of Hg²⁺ or Cd²⁺ with certain lipid classes. A biomimetic liposome system was developed that contained four major lipid classes of erythrocyte membranes (zwitterionic lipids: phosphatidylcholine and phosphatidylethanolamine; negatively charged: phosphatidylserine and neutral: cholesterol). In contrast to Hg²⁺, which preferentially bound to the negatively charged phosphatidylserine compared to the zwitterionic components, Cd²⁺ bound stronger to the two zwitterionic lipids. Thus, the observed distinct differences in the binding affinity of Hg²⁺ and Cd²⁺ for certain lipid classes together with their known effects on membrane properties represent an important first step toward a better understanding the role of these interactions in the chronic toxicity of these metals.     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

Ce^3+和La^3+对人工老化沙葱种子活力及生理特性的影响

以未老化和人工老化后的沙葱(Allium mongolicum Regel.)种子为材料,采用氯化铈(Ce3+)和氯化镧(La3+)浸种,测定种子萌发和生理指标,探讨Ce3+和La3+浸种对种子萌发、老化种子活力和生理特性的影响。结果显示:(1)在老化0~5 h时,Ce3+和La3+处理可显著促进沙葱种子萌发,提高种子活力;在老化5 h后,Ce3+和La3+处理对种子萌发无明显促进作用。(2)在老化0~15 h时,Ce3+和La3+处理的沙葱种子中抗氧化酶活性和抗坏血酸(AsA)含量提高,其超氧阴离子自由基(O2-·)产生速率、过氧化氢(H2O2)含量和丙二醛(MDA)含量显著降低;在老化15 h后,Ce3+和La3+处理的种子抗氧化酶活性提高、AsA含量降低,O2-·产生速率和MDA含量提高。(3)在老化5 h时,沙葱种子呼吸速率发生跃变达到最大,Ce3+和La3+处理显著降低了种子呼吸速率。(4)Ce3+和La3+处理在老化0~5 h时提高了沙葱种子超弱发光(UWL)强度,但在老化5 h后沙葱种子的UWL强度降低。研究认为,在沙葱种子人工老化初期,Ce3+和La3+浸种处理可以诱导增强种子抗氧化酶活性和提高AsA含量,有效清除因老化产生积累的过量活性氧(ROS),减轻过氧化伤害,提高种子活力;种子老化中后期,其内部ROS产生与清除系统发生紊乱,加剧了ROS对种子结构的损伤,Ce3+和La3+浸种处理的缓解效应丧失。     

Ganglioside GM3 modulates conformation of reconstituted ca2+-ATPase

Using steady-state fluorescence and nanosecond time-resolved fluorescence techniques, the ca²⁺ ATPase conformational changes induced by ganglioside GM3 were studied with different quenchers. The results showed that GM3 could significantly increase the lifetime of intrinsic fluorescence of Ca²⁺-ATPase reconstituted into proteoliposomes, and could also weaken the intrinsic fluorescence quenching by KI or hypocrellin B, HB. Furthermore, by using quenching kinetic analysis of the time-resolved fluorescence, in the presence of GM3, the quenching constant (K_3V) and quenching efficiency were significantly lowered. The obtained results suggest that the oligosaccharide chain and the ceramide moieties of the GM3 molecule could interact with its counterparts of the ca²⁺-ATPase respectively, thus change the conformation of the hydrophobic domain of the enzyme, making the tryptophan residues in different regions shift towards the hydrophilic-hydrophobic interface, and hence shorten the distance between the hydrophilic and the hydrophobic domains, making the enzyme with a more compact form exhibit higher enzyme activity. Project supported by the State Key Laboratory of Biomacromolecules.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

Bivalent Metal Ions Modulate Cd2+ Effects on Isolated Rat Liver Mitochondria

We have studied Cd²⁺-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd²⁺] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd²⁺ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd²⁺ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO₃ or NH₄NO₃ medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd²⁺ effects on mitochondria treated in sequence with 100 M of Ca²⁺, Sr²⁺, Mn²⁺ or Ba²⁺(Me²⁺) and 7.5 M RR, as well as the alterations in Cd²⁺ action on the uptake of ¹³⁷Cs⁺ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca²⁺ or RR. The evidence obtained indicate that Ca²⁺ exhibits a synergestic action on all Cd²⁺ effects examined, whereas Sr²⁺ and Mn²⁺, conversely, are antagonistic. In the presence of RR, the Cd²⁺ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd²⁺, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd²⁺-stimulated swelling and ¹³⁷Cs⁺ (analog of K⁺) uptake in the acetate media. For the first time, we have shown that Cd²⁺-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.     

Immunological crossreactivity between the retinal Na+-Ca2+,K+ and the cardiac Na+-Ca2+ exchanger proteins

We have used a series of monoclonal antibodies (mAbs) to determine the degree of microscopic structural homology between the retinal Na⁺-Ca²⁺, K⁺ and the cardiac Na⁺-Ca²⁺ exchange proteins. Sets of mAbs were raised separately to partially purified preparations of either the retinal or the recombinant myocardial exchanger. Each panel of mAbs was then screened for crossreactivity with the respective heterologous exchanger using enzyme-linked immunoassay and immunoblotting techniques. Out of 43 anti-retinal exchanger mAbs, we found 3 detecting the cardiac exchanger on immunoblots, while 4 out of 36 anti-cardiac exchanger mAbs reacted with the retinal exchanger. The strength of the crossreactions was generally weak and suggested that only low affinity epitopes were available on the heterologous proteins. For two crossreacting anti-retinal mAbs the apparent binding affinities to the cardiac exchanger were lower by more than two orders of magnitude. The overall low degree of epitope sharing among the two sets of mAbs confirms that in spite of their obvious functional and topological similarities, microscopic structural homologies between the two proteins are scarce.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Ce4+-stimulated ion fluxes are responsible for apoptosis and taxol biosynthesis in suspension cultures of Taxus cells

Ion fluxes across the plasma membrane activated by 1 mM Ce⁴⁺, cell apoptosis and taxol biosynthesis in suspension cultures ofTaxus cuspidata were studied. The extracellular pH sharply decreased upon the addition of 1 mM Ce⁴⁺, then increased gradually and exceeded the initial pH value over a time period of 12 h. The extracellular Ca²⁺ concentration decreased within the first 3 h after the addition of Ce⁴⁺, then gradually decreased to one third of initial value in control at about 72 h and remained unchanged afterwards. Experiments with an ion channel blocker and a Ca²⁺-channel blocker indicated that the dynamic changes in extracellular pH and the Ca²⁺ concentration resulted from the Ce⁴⁺-induced activation of H⁺ uptake and Ca²⁺ influx across the plasma membranevia ion channels. A pretreatment of the ion channel blocker initiated Ce⁴⁺-treated cells to undergo necrosis, and the prior addition of the Ca²⁺-channel blocker inhibited Ce⁴⁺-induced taxol biosynthesis and apoptosis. It is thus inferred that H⁺ uptake is necessary for cells to survive a Ce⁴⁺-caused acidic environment and is one of the mechanisms of Ce⁴-induced apoptosis. Furthermore, the Ca²⁺ influx across the plasma membrane mediated both the Ce⁴⁺-induced apoptosis and taxol biosynthesis.     

Interaction between Tubulin and Na2+,K2+-ATPase in Brain Stem Neurons

Functionally active Na²⁺,K²⁺-ATPase isozymes containing three types of the catalytic subunits (1, 2, and 3) were obtained from calf brain by two methods: selective removal of contaminating proteins according to Jorgensen (1974) and selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). All preparations were characterized with respect to ouabain-inhibition constants. The presence of the cytoskeleton protein tubulin (3 isoform) in the high-molecular-weight complex of Na²⁺,K²⁺-ATPase 31 isozyme from brain stem axolemma and the junction between Na²⁺,K²⁺-ATPase 3 subunit and tubulin 3 subunit are shown for the first time.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Hg2+、Cd2+和Cu2+对菹草光合系统及保护酶系统的毒害作用

研究了Hg2 、Cd2 、Cu2 对高等水生沉水植物菹草光合系统及保护酶系统的毒害作用。结果表明 :3种离子均使菹草叶片叶绿体自发荧光强度、叶绿素含量、光合速率降低。Hg2 、Cd2 、Cu2 对菹草保护酶系统存在不同的影响 ,短时间低浓度条件下 ,诱导超氧化物歧化酶 (SOD )、过氧化氢酶 (CAT)、过氧化物酶(POD)活性上升 ,随着污染时间的延长、污染浓度的增加 ,酶活性下降 ,其中SOD活性上升持续时间最长 ,下降最慢。 3种离子均使菹草可溶性蛋白含量减少。Hg2 、Cu2 毒性较强 ,Cd2 毒性较弱。Hg2 、Cu2 对菹草的致死浓度为 0 .5～ 1mg/L ,Cd2 为 1～ 2 .5mg/L     

Neutral organic solute effects on the activity of the plasma membrane Ca2+-ATPase

We have compared effects of dimethylsulfoxide (Me₂SO) and two polyols on the Ca²⁺-ATPase purified from human erythrocytes. As studied under steady-state conditions over a broad solute concentration range and temperature, Me₂SO, glycerol, and xylitol do not inhibit the Ca²⁺-ATPase activity; this is in contrast to numerous other organic solutes that we have investigated. Under specific experimental conditions, Me₂SO (but not glycerol) substantially increases Ca²⁺-ATPase activity, suggesting a possible facilitation of enzyme oligomerization. The activation is more pronounced at low Ca²⁺ concentrations. In contrast to glycerol, Me₂SO shows no protective effect on enzyme structure as assessed by determining residual Ca²⁺-ATPase activity after exposing the enzyme to thermal denaturation at 45°C. Under these conditions several other organic solutes strongly enhance the denaturating effect of temperature. Because of the temperature dependence of its effect on the Ca²⁺-ATPase activity we believe that Me₂SO activates the Ca²⁺-ATPase by indirect water-mediated interactions.     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

The binding of ATP to the catalytic and the regulatory site of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum

Two kinds of ATP binding sites were found on the ATPase molecule in deoxycholic acid-treated sarcoplasmic reticulum. One was the catalytic site (1 mol/mol active site) and its affinity was high. Upon addition of Ca²⁺, all the ATP bound to the catalytic site disappeared at 75 mM KCl, while a significant amount of ATP remained bound to the site at 0–2 mM KCl. The latter binding was found to be due to the formation of a slowly exchanging enzyme-ATP complex, which is in equilibrium with phosphoenzyme + ADP. The other binding site was the regulatory one (1 mol/mol active site) and its affinity was low, changing only insignificantly upon addition of Ca²⁺. The ATP binding to the regulatory site shifted the equilibrium between the slowly exchanging complex and EP toward EP.     

亚硒酸钠对巨噬细胞内游离Ca2+和Mg2+的影响

用单细胞阳离子测定系统研究了SeO^2-₃对巨噬细胞内游离Ca²⁺和Mg²⁺的影响.实验结果表明:SeO^2-₃高于10^-4mol/L时,有显著的细胞毒性.SeO^2-₃对细胞的毒性作用使细胞内游离Ca²⁺和Mg²⁺的浓度升高但Ca²⁺浓度的升高速率比Mg²⁺快.还有,高于10^-4mol/L的SeO^2-₃对红细胞膜上的Ca²⁺-ATP酶活性有明显抑制作用.