Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

盐胁迫对二穗短柄草幼苗生长及不同器官中盐离子稳态的影响

采用4种浓度的NaCl溶液(50、100、150、200 mmol/L)处理二穗短柄草和拟南芥(对照)幼苗,测定不同浓度盐胁迫下2种植物幼苗的生长指标和离子分布,以探讨二穗短柄草在盐胁迫下主要阳离子平衡机制.结果表明:(1)盐胁迫显著抑制二穗短柄草根叶的生物量积累.(2)根冠比数据显示,在盐胁迫条件下二穗短柄草能够更好地维系地下部分的生物量积累.(3)在4种浓度盐胁迫下,二穗短柄草叶中Na+含量低于根系,而且K+、Cl-含量和K+/Na+比值始终高于根系,说明在二穗短柄草中Na+从地下到地上的转运受到抑制,但对Cl-的转运缺乏有效的调控.(4)回归分析发现,盐胁迫下二穗短柄草和拟南芥根部Na+与K+含量变化呈正相关关系,而在叶部则不相关,说明二穗短柄草和拟南芥Na+与K+在根部具有相同的离子通道,而在叶部却具有各自独立的转运途径.     

Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na⁺ + K⁺)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C₁₂E₈). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K⁺-phosphatase or (Na⁺ + K⁺)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the α- and β-subunits. The (Na⁺ + K⁺)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis α-toxin-Sepharose columns. The data suggest that the α-subunit of (Na⁺ + K⁺)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.     

Mathematical modelling of ATP, K and Na interactions with (Na + K)-ATPase occurring under equilibrium conditions

The controlling effect of ATP, K⁺ and Na⁺ on the rate of (Na⁺ + K⁺)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.

1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.

2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K⁺ and Na⁺ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.

3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)₂ complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K⁺ and Na⁺ were absent or K⁺ and K⁺ plus Na⁺, respectively, were present in the experiments.

4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na⁺ + K⁺)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.

互花米草NHX2基因的克隆与功能鉴定

NHX2属于CPA1基因家族,编码Na~+/H~+逆向转运蛋白,控制液泡膜中活性K~+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。     

Studies on (Na + K)-activated ATPase XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na⁺ + K⁺)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na⁺ + K⁺)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na⁺ + K⁺)-ATPase activity. These results indicate that cholesterol is not essential for (Na⁺ + K⁺)-ATPase activity.     

高盐生境中乌拉尔甘草盐离子分泌与积存格局的研究

采用植物水培方法,以乌拉尔甘草为研究材料,用不同浓度(0、80、160、320mmol·L~(-1))NaCl溶液胁迫处理乌拉尔甘草幼苗3周后,分析其叶片表面盐离子(K~+、Ca~(2+)、Na+)分泌速率的差异,并采集盐化低地草甸重盐土生境中2年生乌拉尔甘草植株,应用ICP-AES测定其不同部位(根、根状茎、茎、老叶和幼叶)中的盐离子(K~+、Na~+、Ga~(2+)、Mg~(2+))含量,探究盐离子在乌拉尔甘草叶片上的分泌格局以及盐离子在植株体内的积存格局,为完善甘草耐盐机理的研究提供依据。结果显示:(1)随着盐胁迫浓度的升高,乌拉尔甘草叶片上K~+、Ca~(2+)、Na+的分泌速率均呈增加趋势,且Na~+的分泌速率远远大于Ca~(2+)和K+的分泌速率。(2)在乌拉尔甘草各部位中,K+的积存量从大到小依次为:幼叶根根状茎茎老叶;Na~+在各个部位的积存量都十分有限,且无论地下部分还是地上部分,差异均不大;Ca~(2+)积存量由大到小依次为:老叶幼叶茎根状茎根,且老叶中Ca~(2+)的积存量显著高于其它部位。研究认为,在重盐碱地生境中,K+主要积存在幼叶中,Ga~(2+)主要积存在老叶中,植株体内各个部位Na~+的积存量很低,乌拉尔甘草表现出明显的拒Na现象;叶片分泌的主要盐离子为Na~+;乌拉尔甘草通过泌盐的方式将Na+排出体外,从而有效降低Na~+在体内的积存,这是其能够在重盐碱地生存生长的重要原因。     

披针叶黄华试管苗适应盐胁迫的渗透调节机制

以披针叶黄华(Thermopsis lanceolata)试管苗为材料,通过组培方法研究其在0、0.2%、0.4%、0.6%、0.8%和1.0%NaCl和Na2SO4胁迫30d后的生长、有机渗透调节物质和无机渗透调节物质(Na+、K+和Ca2+)含量的变化,以探讨其耐盐性机制。结果显示:(1)随NaCl和Na2SO4胁迫浓度的增加,披针叶黄华试管苗叶片脯氨酸和可溶性糖含量均显著持续增加,且NaCl胁迫下脯氨酸上升的幅度均大于相同浓度Na2SO4胁迫下的增幅,而可溶性糖上升的幅度却小于相同浓度Na2SO4胁迫下的幅度;可溶性蛋白含量随NaCl浓度的增大呈先升高后降低的趋势,但随Na2SO4浓度的增加呈持续上升的趋势。(2)随NaCl和Na2SO4浓度的增加,披针叶黄华试管苗Na+含量呈增加趋势且各处理均显著高于对照,Ca2+含量和叶片K+含量却呈逐渐减少趋势且各处理均显著低于对照,而根系K+含量呈先降后升的趋势;Na2SO4胁迫下披针叶黄华试管苗叶片Na+含量上升幅度以及K+和Ca2+含量下降幅度均明显低于相同浓度NaCl胁迫组;而Na+/K+和Na+/Ca2+比值随NaCl和Na2SO4浓度增加而升高;NaCl胁迫下,叶片Na+/K+和Na+/Ca2+高于相同浓度Na2SO4胁迫下的比值,而根系Na+/K+和Na+/Ca2+却低于相同浓度Na2SO4胁迫下的比值。研究表明,盐胁迫下,披针叶黄华试管苗通过抑制叶片中Na+积累并增加可溶性糖和可溶性蛋白含量,在根系中维持较高K+和Ca2+含量以及较低水平Na+/K+和Na+/Ca2+比,以降低披针叶黄华细胞渗透势来适应盐渍环境;披针叶黄华对NaCl胁迫的调节能力弱于Na2SO4。     

NaCl胁迫对圆柏幼苗生长和离子吸收及分配的影响

以当年生圆柏幼苗为实验材料,采用温室调控盆栽土培法研究了不同浓度NaCl(0、100、200、300mmol·L-1)胁迫21d对其生长情况及不同器官(根、茎、叶)中K~+、Na~+、Ca~(2+)和Mg~(2+)的吸收和分配的影响,以探讨圆柏幼苗对盐环境的生长适应性及耐盐机制。结果表明:(1)随着NaCl胁迫浓度的增加,圆柏幼苗生长,包括株高、地径、相对生长量以及生物量的积累均呈下降趋势,而其根冠比却增加。(2)在各浓度NaCl胁迫处理下,圆柏幼苗根、茎、叶中Na~+含量较对照均显著增加,而且叶中Na~+含量显著高于茎和根,叶中Na~+含量是根中的5倍。(3)随着NaCl胁迫浓度的升高,圆柏幼苗各器官中K~+、Ca~(2+)和Mg~(2+)含量以及K~+/Na~+、Ca~(2+)/Na~+及Mg~(2+)/Na~+比值均呈下降趋势。(4)在NaCl胁迫条件下,圆柏幼苗根系离子吸收选择性系数SK,Na、SCa,Na、SMg,Na显著提高,茎、叶离子转运选择性系数SCa,Na、SMg,Na则逐渐降低,叶中离子转运选择性系数SK,Na则随着NaCl胁迫浓度的升高显著降低,大量Na~+进入地上部,减缓了盐胁迫对根系的伤害。研究认为,圆柏幼苗的盐适应机制主要是通过根系的补偿生长效应及茎、叶对Na~+的聚积作用来实现的,同时也与根对K~+、Ca~(2+)、Mg~(2+)的选择性运输能力增强和茎、叶稳定的K~+、Ca~(2+)、Mg~(2+)的选择性运输能力有关。     

盐渍土壤大果沙枣树主要矿质阳离子的吸收和分配特征

为探明大果沙枣树体矿质离子渗透调节机制,比较分析了盐渍化生境中1～12a生树的根、枝和叶部主要矿质阳离子的吸收、分配特征.结果 表明:(1)大果沙枣树体内Ca2+的积累量最高(13.79 g/kg),K+次之(5.92 g/kg),Na+最低(1.00 g/kg);随着树龄的增大,大果沙枣根部的Na+以及枝和叶部的K+...     

Physiological characterization of four model Lotus diploid genotypes: L. japonicus (MG20 and Gifu), L. filicaulis, and L. burttii under salt stress

The genus Lotus comprises a heterogeneous group of annual and perennial species. Lotus japonicus (with MG20 and Gifu ecotypes) has been adopted as one of the model legumes in genetic and genomic studies. Other Lotus species, such us Lotus burttii and Lotus filicaulis, have also been used in genetic and genomic studies because of their capacity to produce fertile progenies in crosses with L. japonicus. In the present work, physiological responses to salt stress in four Lotus genotypes were evaluated on the basis of growth and associated parameters, such as photosynthesis, ions, relative water content, oxidative damage and antioxidant system responses, using two NaCl levels applied by acclimation for up to 28 and 60 d. Growth responses varied with plant developmental stage in the four Lotus genotypes. L. japonicus MG20 was found to be a salt-tolerant genotype, mainly when exposed to salt stress at the young plant stage. The capacity of Lj MG20 to sustain growth under salt stress was correlated with enhancement of Superoxide dismutase and Glutathione reductase activities, as well as with increases in total and reduced glutathione content and lower Na⁺ accumulation in leaves. These results suggest that enhancement of antioxidant responses in Lj MG20 contributed to improve salt stress tolerance at early stages. On the other hand, after long-term high NaCl stress treatment, L. filicaulis exhibited lower biomass reduction, lower oxidative damage and Na⁺ accumulation in leaves than the control treatment; hence, this genotype was considered salt-tolerant. These apparently ambiguous results remark that salt tolerance, as a development-related process, was differentially expressed among the Lotus genotypes and depended on stress duration and plant phenological stage.     

In vivo modification of Na,K-ATPase activity in Drosophila

We have constructed and characterized transgenic Drosophila lines with modified Na⁺,K⁺-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na⁺,K⁺-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (α^D369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system we have limited expression of α transgenes to cell types that normally express a particular Drosophila Na⁺,K⁺-ATPase β (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.     

长苞香蒲对人工盐碱湿地Na+和K+的吸收与转运特征

为揭示长苞香蒲(Typha domingensis)对盐生湿地生态系统中Na⁺和K⁺的吸收与转运特征,探讨长苞香蒲对盐生湿地的生态修复效果,该研究采用人工模拟盐生湿地的方法,设置CK(对照)、T1(浇灌100 mmol·L^-1盐水)、T2(浇灌200 mmol·L^-1盐水)及T3(浇灌300 mmol·L^-1盐水)4种不同盐浓度的人工湿地生态系统,并分别于5月5日(开始盐胁迫处理,S0)、5月30日(S1)、6月30日(S2)和7月30日(S3)测量其株高和干重、植株地上与地下部分Na⁺和K⁺的含量以及底泥和水体中Na⁺和K⁺的含量以分析长苞香蒲对盐碱湿地的脱盐作用。结果表明：(1)各处理的长苞香蒲的株高和干重随着处理时间的延长呈增加趋势,但与CK相比,各处理生长量随盐浓度升高出现下降趋势。(2)高浓度盐处理(T3)使长苞香蒲的地上部分和地下部分的Na⁺分别增加了2.5...     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

Genetic Na channelopathies and sinus node dysfunction

Voltage-gated Na⁺ channels are transmembrane proteins that produce the fast inward Na⁺ current responsible for the depolarization phase of the cardiac action potential. They play fundamental roles in the initiation, propagation, and maintenance of normal cardiac rhythm. Inherited mutations in SCN5A, the gene encoding the pore-forming α-subunit of the cardiac-type Na⁺ channel, result in a spectrum of disease entities termed Na⁺ channelopathies. These include multiple arrhythmic syndromes, such as the long QT syndrome type 3 (LQT3), Brugada syndrome (BrS), an inherited cardiac conduction defect (CCD), sudden infant death syndrome (SIDS) and sick sinus syndrome (SSS). To date, mutational analyses have revealed more than 200 distinct mutations in SCN5A, of which at least 20 mutations are associated with sinus node dysfunction including SSS. This review summarizes recent findings bearing upon: (i) the functional role of distinct voltage-gated Na⁺ currents in sino-atrial node pacemaker function; (ii) genetic Na⁺ channelopathy and its relationship to sinus node dysfunction.     

西伯利亚白刺质膜Na+/H+逆向转运蛋白基因NsSOS1的分离及表达分析

采用同源克隆技术分离了西伯利亚白刺(Nitraria sibirica)质膜Na~+/H~+逆向转运蛋白基因NsSOS1,并对其在不同胁迫条件下的表达特性进行了分析。NsSOS1包含3 516bp开放阅读框(ORF),编码1 171个氨基酸,蛋白分子量为128.34kD。生物信息学分析显示,NsSOS1包含12个跨膜结构域,具有植物SOS1蛋白的保守结构域。系统发育分析表明,NsSOS1与其他植物质膜Na~+/H~+逆向转运蛋白处于同一个次级分化群,与锦葵科海滨锦葵KvSOS1亲缘关系较近。实时荧光定量RT-PCR分析显示,NsSOS1基因在西伯利亚白刺的根和叶中表达量较高;其表达受到非生物胁迫(NaCl、低温、干旱)和外源激素(MeJA和GA)的诱导,表明NsSOS1基因在西伯利亚白刺抵御逆境胁迫过程中发挥重要作用。     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

外源Ca2+和IBA对NaCl胁迫下能源植物杂交狼尾草幼苗生长的影响

以能源植物杂交狼尾草(Pennisetum americanum×P.purpureum)为实验材料,在NaCl胁迫条件下用外源IBA(100 mg/L),CaCl_2(浓度分别为0、1、2、5 mmol/L)处理杂交狼尾草幼苗,处理3周后测定植物的存活率、鲜重、干重、株高、生根数和地上部分、地下部分的离子含量。结果表明,经过IBA溶液预处理的杂交狼尾草幼苗的存活率、鲜重、干重、株高、生根数明显高于未处理的幼苗;在NaCl胁迫下,随着外源Ca~(2+)浓度的升高,杂交狼尾草幼苗的存活率、鲜重、干重、株高、生根数以及Ca~(2+)含量都明显升高并在CaCl_2浓度为2 mmol/L时达到最大值;随着外源Ca~(2+)浓度的升高,Na~+含量、Na~+/K~+降低,当CaCl_2的浓度为2mmol/L时,Na~+含量、Na~+/K~+最低。以上结果表明外源Ca~(2+)和IBA对NaCl胁迫下杂交狼尾草幼苗生长有促进作用,可以缓解NaCl胁迫对杂交狼尾草幼苗生长的抑制作用,提高杂交狼尾草幼苗在NaCl胁迫下的成活率;缓解盐害的最适的Ca~(2+)浓度为2mmol/L。