Mg2+和Na+对多核酶系统体外切割反应的影响

为了进一步了解2价Mg^2＋和1价Na+存在与否的情况下,多核酶系统对底物RNA的切割效率,构建了pGEM Coat′A,pGEM Coat′A196Rz 质粒和pGEM MDR1靶质粒．通过用SP6/T7转录试剂盒在体外转录RNA, 在无细胞系统进行切割反应,反应产物通过6％变性聚丙烯酰胺凝胶电泳,干胶、X光片曝光自显影,利用Image J 生物图像分析软件分析. 结果表明,多核酶系统的切割效率依赖于二价Mg^2＋的浓度,切割产物随Mg^2＋浓度的增加而增加,而且具有反应时间的依赖性. 在Na+浓度低于200 mmol/L且单独存在时,没有切割产物生成．相反,在Na⁺和Mg^2+共存时,表现出Na⁺抑制Mg2+诱导的切割活性,切割效率明显低于Mg²⁺单独存在时的结果.这些结果提示,在生理环境下,Mg²⁺对于多核酶系统对底物的切割反应是必需的,而Na⁺则不是．     

SAM-V核糖开关RNA体外转录体系优化

为了提高RNA体外转录效率,选取SAM-V核糖开关晶体结构模板对RNA体外转录体系进行优化。结果表明:目的RNA相对百分含量在反应时间和Mg2+添加浓度分别为8h和85m M/L时达到最高值,优化体系较标准体系目的RNA相对百分含量提高了一倍。我们的工作为下一步准备SAMV核糖开关晶体打下了基础。     

专一切割苹果锈果类病毒多体自切割核酶的克隆和转录物的体外活性测定

将苹果锈果类病毒的1个14nt的靶序列连接在锤头型核酶的3′末端,构成自切割核酶。经人工合成和PCR扩增,克隆在转录载体pGEM7zf（＋）的XhoⅠ-Hind Ⅲ位点。利用限制酶Xho I与SalI的连接,消失其识别位点序列,将自切割核酶片段插入到重组质粒中,经连续5次亚克隆,分别获得2、4、6、8、10和12拷贝的多体自切割核酶。在T7RNA聚合酶作用下,线性化重组质粒转录的多体自切割核酶通过内部的顺式切割释放出较多数量的核酶分子,提示在转录水平能够提高核酶转录物的浓度。用相同摩尔浓度的单体和12体自切割核酶分别对32P标记的靶ASSVd进行反式切割,核酶与靶RNA摩尔浓度比为1：1。放射自显影结果表明：多体自切割核酶对靶ASSVd的切割效率明显高于单体自切割核酶。我们推测多体自切割核酶在体内系统中可能具有更好的应用价值。     

Mg2+浓度对细胞融合效果的影响

目的:细胞融合是细胞生物学中一种常用的技术,有着广泛的应用,如单克隆抗体制备,核质研究,疫苗研发等.其中,聚乙二醇(PEG)化学融合是最为常用的一种细胞融合技术,影响PEG化学细胞融合效果的因素有很多,但是对一些具体因素的研究的不是很全面.本文旨在为了更全面的了解PEG诱导的化学细胞融合的影响因素,优化融合条件,以此扩大PEG化学融合应用范围.方法:以鸡血血红细胞为材料,通过调节已有的Hanks融合液中镁离子浓度,比较各实验组以及对照组的细胞融合率,探究了Mg2+浓度对细胞融合效果的影响,确定了为提高细胞融合效率应使用的Mg2+浓度区间.结果:可以在原有的Hanks配方的基础上,调节Mg2+浓度至10 mmol/L-20 mmol/L这个范围,细胞融合率较大.结论:Mg2+对细胞融合有一定影响,通过调节Mg2+浓度至上述合适区间可以达到较高的细胞融合率,从而为PEG化学融合提供了一种优化方案.     

U6嵌合型抗caspase-3核酶的体外及体内活性鉴定

为探讨caspase-3基因在细胞凋亡中的作用,及不同表达核酶的载体在体内的表达效果,本研究对比了3种表达核酶的真核质粒,包括RNA聚合酶Ⅱ启动的p1.5RZ107(自我剪切)和pRZ107及RNA聚合酶Ⅲ启动的嵌合于U6中的pU6RZ107在体外和在肝细胞BRL-3A内的活性,以期获得细胞内切割活性较高的的核酶载体方面的信息.结果显示,具有自我剪切功能的质粒p1.5RZ107在体外切割靶RNA的效率最高,几达80%;而体内caspase-3在RNA,蛋白水平及蛋白功能活性上均显著下降,证明核酶在体内均可有效地表达并切割底物,以pU6RZ107切割效率最高,约达65%,pRZ107次之,p1.5RZ107最低.结果表明,U6嵌合型核酶pU6RZ107体内可有效地表达核酶及下调靶RNA水平,这不仅为探讨caspase-3在凋亡途径中的作用,也可为今后的基因治疗提供研究基础.     

结核分枝杆菌ICL mRNA特异性10-23脱氧核酶切割 活性的鉴定及其切割特点

为鉴定结核分枝杆菌异柠檬酸裂合梅(ICL)特异性的10-23DRz在无细胞体系切割ICL mRNA的活性,并探讨其在不同条件下以及联合应用时对靶mRNA的切割特点,采用计算机软件模拟ICL mRNA的二级结构,据此选择适合的待切割靶点并设计针对相应靶点的特异性10-23DRz（DZ1～DZ5）.PCR法扩增获得icl基因并克隆入质粒pET32a+.采用T7 RNA聚合酶体外转录法获取ICL全长mRNA后分别用DZ1～DZ5在无细胞体系中对ICL mRNA进行切割,切割产物经变性聚丙烯酰胺凝胺电泳后用银染法鉴定各DRzs的活性.选择切割活性最强的DZ4考察不同10-23DRz剂量、不同反应时间、不同镁离子浓度条件下及不同错配或突变10-23DRz的切割特点.联合应用DZ1、DZ4及DZ5在无细胞体系中对ICL mRNA进行切割,检测10-23DRz联合应用对切割效率的影响.结果表明,DZ1、DZ3、DZ4及DZ5可在无细胞体系中有效地切割ICL mRNA,其切割效率在30.8%～64.5%之间.对DZ4切割活性的检测发现,其对靶mRNA的切割具有剂量和时间的依赖性;在2～20 μmol/L范围内,DZ4的切割活性与Mg2＋浓度呈正相关;DZ4单侧底物结合臂上含一个不与靶mRNA配对的碱基时其切割效率大大降低,两侧底物结合臂上各含一个不配对的碱基或活性中心域第6位出现碱基突变（G→C）时,DZ4完全丧失切割活性.联合应用2种或2种以上10-23DRz可显著增强对底物RNA的切割效率.10-23 DRz特异、有效地切割结核分枝杆菌ICL全长mRNA并显示一定的叠加效应,有望用于抗结核分枝杆菌潜伏感染的基因治疗.     

纳米TiO2、ZnO、SiO2对剑尾鱼肝组织中Na+/K+-ATP酶活性的影响

采用浸浴法研究了氧化纳米颗粒TiO2、ZnO、SiO2对剑尾鱼(Xiphophorus helleri)肝中Na+/K+-ATP酶活性的影响。结果表明:纳米TiO2处理组,高浓度(5 mg/L、10 mg/L)组表现为抑制作用,其中5 mg/L处理组Na+/K+-ATP酶的活性与对照组无显著差异(p>0.05),10 mg/L处理组中Na+/K+-ATP酶的活性显著低于对照组中酶的活性(p<0.05)。低浓度组(0.1 mg/L、1 mg/L)则表现为先诱导后抑制,除0.1 mg/L组在暴露1 d后与对照组有显著差异外(p<0.05),其余组与对照组均无显著差异(p>0.05)。纳米ZnO、SiO2处理组(0.1 mg/L、10 mg/L)在暴露1 d后,肝中Na+/K+-ATP酶的活性均比对照组高,随着暴露时间增加至20 d,Na+/K+-ATP酶活性下降,且显著低于对照组(p<0.05)。3种纳米颗粒的浓度为0.1 mg/L时,对暴露后1 d剑尾鱼肝中的Na+/K+-ATP酶活性的影响均为诱导作用,诱导大小顺序为ZnO>TiO2>SiO2;随着暴露时间的增加至10 d,纳米TiO2、ZnO、SiO2处理组对Na+/K+-ATP酶活性均表现出抑制作用。     

NaCl和Na2CO3胁迫对桑树幼苗生长和光合特性的影响

以1年生“青龙桑”幼苗为试验材料,研究了中性盐(NaCl)和碱性盐(Na2CO3)胁迫下桑树幼苗的生长和叶片光合特性.结果表明:盐胁迫明显降低了桑树幼苗的株高、叶片数、生物量和叶片的光合能力.随着Na+浓度的增加,桑树叶片的气孔导度、蒸腾速率、净光合速率、实际光化学效率、电子传递速率和光化学猝灭系数明显降低,过剩光能以非光化学猝灭形式耗散的比例增加,桑树叶片的光能转化效率和光合能力下降.在Na+浓度＜150 mmol·L-1时,桑树幼苗的光合能力和生长受到的抑制较小,通过增加根冠比进一步适应盐胁迫,但这种保护机制随着盐浓度的增加逐渐降低.在Na2CO3胁迫下,＞50 mmol·L-1 Na+浓度对桑树的生长和光合能力表现出较强的抑制作用,并随Na+浓度的增加,抑制程度加大.在NaCl＜ 150mmol·L-1时,桑树的光合能力主要依赖植株形态和光合代谢双重途径适应中性盐逆境,而在NaC1浓度＞150 mmol·L-1和碱性盐胁迫下,其主要依赖光合代谢来适应逆境.     

连续两个生长季大气CO2浓度升高对银杏希尔反应活力和叶绿体ATP酶活性的影响

近年来,随着温室气体浓度不断上升,有关CO2浓度升高对植物影响的研究已取得一定进展,但CO2浓度升高对植物光合作用的影响需要从生理生化水平上进一步深入的研究.以沈阳城市森林树种银杏(Ginkgo biloba L.)为研究对象,利用开顶式气室研究连续两个生长季大气CO2浓度升高对银杏光合特性的影响.结果表明,在大气CO2浓度为700μmol·mol-1条件下,与对照相比,第1个生长季CO2处理的银杏叶片净光合速率极显著增加(P<0.01),希尔反应活力极显著增大(P<0.01)、Ca2+/Mg2+-ATP酶活性显著(P<0.05)或极显著增强(P<0.01)、光合产物淀粉的含量极显著增多(P<0.01);第2生长季CO2处理的银杏叶片净光合速率显著增加(P<0.05),希尔反应活力在通气60d时极显著(P<0.01)增大,Ca2+/Mg2+-ATP酶活性在处理30d时显著降低(P<0.05),淀粉含量增多.与第1个生长季相比,第2个生长季CO2处理的银杏叶片净光合速率降低,希尔反应活力减小,Ca2+/Mg2+-ATP酶活性减弱,叶绿素含量增多,淀粉含量减少.试验中出现了光合适应现象.     

Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures.

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4, 1518-1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34-3.5 and 3.3-0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.     

The hammerhead cleavage reaction in monovalent cations

Recently, Murray et al. (Chem Biol, 1998, 5:587-595) found that the hammerhead ribozyme does not require divalent metal ions for activity if incubated in high (> or =1 M) concentrations of monovalent ions. We further characterized the hammerhead cleavage reaction in the absence of divalent metal. The hammerhead is active in a wide range of monovalent ions, and the rate enhancement in 4 M Li+ is only 20-fold less than that in 10 mM Mg2+. Among the Group I monovalent metals, rate correlates in a log-linear manner with ionic radius. The pH dependence of the reaction is similar in 10 mM Mg2+, 4 M Li+, and 4 M Na+. The exchange-inert metal complex Co(NH3)3+ also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.     

Condensation of the chromatin gel by cations

Calf thymus chromatin gel, containing strongly bound nonhistone proteins, was used to study the effect of easily removable and tightly bound cations on the condensation of chromatin. The chromatin volume was found to be linearly dependent on the reciprocal square root of the concentration of easily removable cations (Tris X H+ + Na+ and Mg2+) except for the initial stages of condensation (up to 7-10 mM monovalent and 0.15-0.2 mM divalent cations). The effect of Mg2+ at the initial stage of condensation was not reproduced by Na+ and vice versa. At higher concentrations the effects of Na+ and Mg2+ were additive. The removal of tightly bound divalent cations by a treatment of the chromatin gel with 1,10-phenanthroline led to an approx. 50% increase in the volume of the chromatin gel, which was maintained at each concentration of easily removable cations. The 1,10-phenanthroline-caused decondensation of the chromatin gel was reversed by Ca2+ but not by Mg2+, Zn2+ and Cu2+. The chromatin gel pretreated with Ca2+ was not further decondensed by 1,10-phenanthroline.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

Interaction of sodium and potassium ions with Na+,K+-ATPase. II. General properties of ouabain-sensitive K+ binding

General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.     

Uridine uptake inhibition in Novikoff hepatoma cells by perturbants of intracellular Ca2+ and K+ levels

The rate of uptake of uridine into the acid-soluble fraction of Novikoff hepatoma cells is inhibited by low concentrations of the ionophores A23187 and gramicidin and other perturbants of intracellular cation levels. Inhibition of uridine uptake by A23187 is dependent on Ca2+ and is reduced by serum and high levels of Mg2+. The effectiveness of A23187 is dependent on the Ca2+/Mg2+ ratio rather than the absolute concentration of either ion. Inhibition of uridine uptake by gramicidin is not significantly affected by serum or divalent cations. Other effectors of monovalent cation flux such as ouabain and valinomycin also inhibit uridine uptake. These results indicate that net uptake of uridine may be influenced by intracellular levels of certain monovalent and divalent inorganic cations.     

An improved method for production of silica from rice hull ash

Biosorption of monovalent ions Na+ and K+, by deactivated protonated yeast (Saccharomyces cerevisiae) at controlled pH, was compared with biosorption of divalent ions Ca2+ and Mg2+ to help to understand the underlying bindingmechanisms. The adsorption for monovalent ions was accompanied by H+ release. Divalent ions were sorbed by proton displacement, and also an additional mode not accompanied by release of H+. The sorption uptake of both monovalent and divalent metal ions increased with pH in the range 3-7 peaking at 6.75. Equilibrium sorption isotherms at pH = 6.75 showed that the totalmaximum biosorptive capacity for metal ions decreased in the following order: Ca > Mg > Na > or = K.     

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mn2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ greater than Co2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ approximately equal to control without cations. Addition of Na2+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 +/- 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 +/- 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, Bmax and affinity were reduced. The addition of 100 microM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K-1). The equilibrium dissociation constant (KD) calculated with these rate constants (K-1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.