金属离子与CaM及Ca~(2+)-Mg~(2+)-ATPase相互作用的荧光光谱和荧光偏振研究

根据Cd~(2+)、Pb~(2+)、Hg~(2+)和Al~(3+)对丹磺酰标记钙调蛋白(D-CaM)的荧先强度、最大发射波长及偏振度的影响来研究它们对CaM及Ca~(2+)-Mg~(2+)-ATPase构象变化的影响.研究发现,无论溶液中是否存在Ca~(2+)-Mg~(2+)-ATPase,Cd~(2+)、Pb~(2+)和Hg~(2+)对D-CaM的荧光最大发射波长、偏振度的影响以Cd~(2+)的最大,Pb~(2+)次之,Hg~(2+)最小,Al~(3+)对D-CaM产生的影响与这三种二价金属离子的并不相同.这证明这几种离子与CaM和Ca~(2+)-Mg~(2+)-ATPase的作用并不遵循同样的机理.     

重金属离子对猪红细胞膜Ca~(2+)-ATP酶的作用

猪红细胞膜Ca~(2+)-ATP酶是一种钙调蛋白(CaM)依赖酶,其活力又依赖巯基的完整性。实验应用Ca~(2+)-ATP酶这一模型体系观察到重金属离子,Pb~(2+)、Cd~(2+)和Hg~(2+)都能替代Ca~(2+),激活CaM,从而激活Ca~(2+)-ATP酶;其最大刺激活力分别为85％、80％和30％,半刺激浓度分别为32、27和0.7μmol/L。当三种重金属离子的浓度增加时,则与Ca~(2+)-ATP酶的巯基结合,抑制酶的活力,Pb2~(2+)、Cd~(2+)和Hg~(2+)的半抑制浓度分别为370、440和2μmol/L。抑制作用为渐进性过程,而刺激作用为即时效应。抑制作用可为巯基化物,特别是二巯基化物所逆转。研究结果提示,CaM可能是重金属中毒最初作用的靶分子,而重金属中毒不仅使CaM“开关”失灵,还可能导致细胞内Ca~(2+)的调节全面失控。     

若干蝙蝠葛碱衍生物对人红细胞膜Ca~(2+)-Mg~(2+)-ATPase活力的影响

本文测定了数种蝙蝠葛碱衍生物对钙调素(CaM)激活的人红细胞膜Ca~(2+)-Mg~(2+)-ATPase活力的影响。结果表明,这些化合物对该酶都有不同程度的抑制作用,其机制表现为竞争性抑制,过量的CaM能完全逆转这些化合物所引起的抑制。当Ca~(2+)-Mg~(2+)-ATPase被胰蛋白酶(trypsin)限制性酶解完全活化后,其活力不再受CaM激活,但仍被这些化合物所抑制。     

粉防已碱与红细胞膜ATPase相互作用的研究

粉防已碱是一种新的钙调蛋白拮抗剂,专一性抑制人红细胞膜上依赖CaM的Ca~(2+)-Mg~(2+)-ATPase。在较高浓度下,它也不同程度地抑制Ca~(2+)-Mg~(2+)-ATPase基本活性、Na~+-K~+-ATPase和Mg~(2+)-ATPase的活性。 除CaM外,不饱和脂肪酸和有限水解均导致膜Ca~(2+)-Mg~(2+)-ATPase的活化,所有这些活化作用被Tet在大约相同的浓度范围内抑制,表明Tet除与CaM结合外,也与膜Ca~(2+)-Mg~(2+)-ATPase结合。 Tet具有抗抵渗溶血的性能,反映了拮抗CaM与药物的膜稳定性间存在相关性。     

稀土元素Pr~(3+)、Nd~(3+)对蚕豆(Vicia feba)叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的影响

本文介绍用二相分配法制备蚕豆叶片原生质膜上的Ca~(2+)·Mg~(2+)-ATPase,用以研究镧系,稀土离子对此酶活性的影响。初步证实Pr~(3+)、Nd~(3+)对依赖于CaM的以及不依赖于CaM的蚕豆叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的抑制不是CaM专一的。     

苄基异喹啉类化合物与钙调素相互作用的荧光光谱研究

 苄基异喹啉类化合物拮抗钙调素(CaM)并抑制依赖CaM的环核苷酸磷酸二酯酶(CaM-PDE)的活力;用荧光测定法可检测它们与钙调素的相互作用。 Ca~(2+)存在下蝙蝙葛碱(D_1)及其衍生物(D_(14))在激发波长340nm处最大发射波长分别为463和455nm,结合CaM后荧光量子产率增加两倍多。它们同CaM的结合均依赖于Ca~(2+)。 本文制备的丹磺酰基CaM(D-CaM)结合Ca~(2+)后荧光最大发射峰值兰移(518→508nm),荧光强度增加22％。在Ca~(2+)存在下小檗胺衍生物E_6能与CaM结合并淬灭Ca~(2+)-D-CaM荧光。 单苄基异喹啉类化合物86040、86045能淬灭CaM的酪氨酸残基的特征荧光。 实验表明,CaM结合D_(14)、E_6、86040和86045的kd值分别为1.3、1.8、9.5和15.7μmol/L,所观察的化合物与CaM的亲和力的大小与它们拮抗CaM,抑制CaM-PDE的酶活力相对应。     

Tb~(3+)作为荧光探针研究钙调蛋白与拮抗药物的相互作用

本文报导以Tb~(3+)作为荧光探针,研究钙调蛋白(CaM)与其拮抗药物分子间相互作用的机制.所用方法简便、快速、灵敏.CaM的内源荧光研究表明,Tb~(3+)类似于Ca~(2+),也能诱导CaM分子构象发生改化,由于CaM分子中Ca~(2+)的第Ⅲ、Ⅳ结合位点上各有一个Tyr线基,如(?)280nm激发,则发生从Tyr向Tb~(3+)的能量转移,从而导致Tb~(3+)在490和545nm处的特征荧光发射大大加强.本文检测了药物分子与Tb~(3+)-CaM结合对该荧光发射的影响.实验表明,TFP与CaM的高亲和位点处于CaM分子C-末端部位,即含第Ⅲ、Ⅳ结构域的半分子上:丙拮抗药物酸枣仁皂甙A则优先结合在含第Ⅰ、Ⅱ的结构域的另一半分子(?).     

马兜铃酸与钙调素相互作用的荧光光谱分析——一种非钙离子依赖性钙调素拮抗剂的研究

本文报道我室近来发现的一种天然钙调素(Calmodulin,CaM)拮抗剂马兜铃酸(Aristolochic acid,ATA)的研究。利用丹磺酰标记的CaM(D-CaM)对马兜铃酸的研究表明,马兜铃酸是一种非钙离子依赖性钙调素拮抗剂,实验测得马兜铃酸与D-CaM结合的解离常数,有Ca~(2+)和无Ca~(2+)情况下分别为70μmol/L、77μmol/L。两种状况下马兜铃酸对D-CaM荧光强度的抑制分别为40％、41％。暗示马兜铃酸主要作用于CaM上Ca~(2+)诱导的疏水区之外。三氟啦嗪(TFP)引起的D-CaM荧光增强可被马兜铃酸明显降低,而TFP在达到马兜铃酸浓度的15倍以上仍未能逆转马兜铃酸对D-CaM荧光强度的降低作用,这为马兜铃酸主要作用于CaM上Ca~(2+)诱导的疏水区以外提供了又一佐证。     

铝与钙调蛋白相互作用的荧光光谱研究

本文研究了铝与钙调蛋白相互作用的荧光光谱。实验证明,Al~3与CaM的结合所引起的构象变化与Ca~(2+)与CaM结合所引起的构象变化既有相同之处,也有不同之处。Al~(3+)在CaM分子上的结合有特异性结合与非特异性结合两种情况。其特异性结合位点可能为2—3个。钙调蛋白的非竞争性拮抗剂酸枣仁皂甙A(JuA)可以继续抑制已被Al~(3+)部分抑制的PDE-CaM的活力。     

Cd~(2+)和Pb~(2+)对花翅摇蚊幼虫的毒性及对三种生物化学标志物的影响

【目的】为了探讨花翅摇蚊ChironomuskiiensisTokunaga对重金属Cd~(2+)和Pb~(2+)胁迫响应。【方法】测定Cd~(2+)和Pb~(2+)对花翅摇蚊4龄幼虫的急性毒性、联合毒性以及体内3种生物化学标志物(CAT、SOD活性和MT含量)的影响。【结果】花翅摇蚊幼虫暴露于重金属Cd~(2+)和Pb~(2+)及混合离子中表现出典型的中毒症状,且24 h和48 h重金属Cd~(2+)致死中浓度LC50显著低于Pb~(2+)的LC50。混合离子Cd~(2+)和Pb~(2+)的联合毒性随着不同配比和作用时间呈现差异性;24hCd~(2+)+Pb~(2+)1︰1配比表现为部分相加作用,Cd~(2+)+Pb~(2+)2︰1和1︰2配比表现为拮抗作用;而作用48 h不同配比联合毒性均为部分相加作用。2种单一和混合重金属Cd~(2+)+Pb~(2+)均不同程度对摇蚊体内SOD和CAT产生抑制作用,而暴露于Cd~(2+)和Pb~(2+)及其混合离子作用下MT含量显著高于对照,且随着时间表现为先增加后降低。【结论】重金属Cd~(2+)和Pb~(2+)对摇蚊幼虫具有急性和联合毒性,体内抗氧化酶系CAT、SOD及体内MT参与了重金属污染物的胁迫的应答响应,并表现出时间-剂量效应,可作为水体环境质量监测的生化标志物。     

Cu~(2+)、Cd~(2+)对中华大蟾蜍肝脏内主要营养物质代谢的影响

实验测定了Cu2+、Cd2+的亚急性毒性处理42 d及在清水中恢复30 d后中华大蟾蜍肝脏中主要营养物质代谢的变化.结果表明,随Cu2+、Cd2+暴露时间的延长,肝糖原含量呈先降后升、总蛋白含量呈先升后降、肝脂肪含量呈持续降低的趋势;在清水中恢复30 d后,肝糖原、总蛋白及脂肪含量均恢复到对照组水平.结果提示,Cu2+、Cd2+暴露过的蟾蜍,在清水中恢复30 d后,其肝脏主要营养物质代谢可以恢复到正常水平.     

不同浓度Hg^2＋对睡莲的毒害影响研究

主要研究了Ｈｇ＾２＋对睡莲的外部形态及叶绿素含量、过氧化物酶活性、硝酸还原酶活性。可溶性蛋白含量等生理指标的影响。结论是：Ｈｇ＾２＋对其外部形态的毒害程度与处理浓度和时间成正相关。１－５ｍｍｏｌ／Ｌ处理使叶绿素含量、硝酸还原酶活性上升,８－１０ｍｍｏｌ／Ｌ处理则下降;１－８ｍｍｏｌ／Ｌ处理过氧化物酶活性随浓度增加而上升,１０ｍｍｏｌ／Ｌ处理则下降,但仍高于对照;可溶性蛋白含量随处理浓度增加呈下降趋     

Cu2+胁迫对2种速生柳幼苗生长及生理特性的影响

以营养溶液中培养的‘苏柳172’(Salix jiangsuensis CL J-172)和垂柳(Salix babylonica Linn)幼苗为材料,分析它们在不同浓度Cu2+溶液(0、20、40、80、100、200、300μmol.L-1)下的生长以及部分生理指标的变化,以明确2种速生柳树用于植物修复的潜力。结果显示:2种柳树幼苗的生物量、叶片色素含量、根系活力以及根系形态的各指标都随Cu2+浓度的增加而显著降低。它们叶片SOD和POD活性以及脯氨酸含量随Cu2+浓度的增加呈先上升后下降的趋势,‘苏柳172’和垂柳中SOD活性在Cu2+浓度为20μmol.L-1时最大,其POD活性分别在Cu2+浓度为100和40μmol.L-1时达到最大,而其脯氨酸含量在Cu2+浓度为200μmol.L-1时达到最大。可见,2种柳树生长均受到Cu2+胁迫的抑制,它们能够在一定程度上通过增加保护酶活性和脯氨酸含量来提高对Cu2+的耐受性,且‘苏柳172’对Cu2+的忍耐力强于垂柳。     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.     

The Ca 2+ pumps and the Na + / Ca 2+ exchangers

The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers     

Mitochondria and Ca(2+) signaling

Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca²⁺ signaling. Mitochondria are capable to sense the levels of cytosolic Ca²⁺ and generate mitochondrial Ca²⁺ responses. Specific mechanisms for both Ca²⁺ uptake and Ca²⁺ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca²⁺ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca²⁺ transport systems and some of the functional consequences of mitochondrial Ca²⁺ uptake     

外源Ca2+对高温强光胁迫下滨梅幼苗的保护效应

以10 mmol/L CaCl2溶液处理滨梅幼苗叶片后,置于培养箱于(40±2)℃高温、光照强度(1 200±50)μmol·m-2·s-1下培养,定期测定有关生理生化指标,以探讨外源Ca2+对高温强光胁迫下滨梅幼苗的保护效应.结果显示:(1)与蒸馏水处理组相比,Ca2+处理使高温强光胁迫下滨梅幼苗叶片的脯氨酸含量显著升高,可溶性糖含量变化不明显,根系活力小幅降低;Ca2+处理有效抑制了高温强光下膜透性的加大,提高和保护了Ca2+-ATPase的活性.(2)采用Ca2+螯合剂EGTA或钙调素拮抗剂TFP对滨梅幼苗叶片同法处理并同条件胁迫时,与Ca2+处理相比,滨梅幼苗的脯氨酸、可溶性糖含量、Ca2+-ATPase活性和根系活力均明显下降,膜透性加大.研究表明,Ca2+处理能提高滨梅幼苗对高温强光的耐受性;Ca2+信号系统参与了胁迫过程中的渗透物质和Ca2+-ATPase活性等的调节.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.     

Mobilizing store Ca(2+) in the presence of La(3+) evokes exocytosis in bovine chromaffin cells

The effect on exocytosis of La(3+), a known inhibitor of plasma membrane Ca(2+)-ATPases and Na(+)/Ca(2+) exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3-3 mM), La(3+) substantially increased histamine-induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd(3+), Eu(3+), Gd(3+), and Tb(3+)), but not several divalent cations. In the presence of La(3+), the secretory response to histamine became independent of extracellular Ca(2+). La(3+) enhanced secretion evoked by other agents that mobilize intracellular Ca(2+) stores (angiotensin II, bradykinin, caffeine, and thapsigargin), but not that due to passive depolarization with 20 mM K(+). La(3+) still enhanced histamine-induced secretion in the presence of the nonselective inhibitors of Ca(2+)-permeant channels SKF96365 and Cd(2+), but the enhancement was abolished by prior depletion of intracellular Ca(2+) stores with thapsigargin. La(3+) inhibited (45)Ca(2+) efflux from preloaded chromaffin cells in the presence or absence of Na(+). It also enhanced and prolonged the rise in cytosolic [Ca(2+)] measured with fura-2 during mobilization of intracellular Ca(2+) stores with histamine in Ca(2+)-free buffer. The results suggest that the efficacy of intracellular Ca(2+) stores in evoking exocytosis is enhanced dramatically by inhibiting Ca(2+) efflux from the cell.