植物钙调素结合蛋白研究进展

钙调素(CaM)作为最重要的一类Ca2 传感蛋白可以通过与其下游CaM结合蛋白(CaMBP)作用而调节细胞的生理功能.因此,对CaMBP的研究是揭示CaM作用机制的重要内容,是探明Ca2 -CaM信号转导系统的关键.该文从CaMBP和CaM的结合特性、植物CaMBP的分布以及植物CaMBP的生物学功能等方面综述了植物CaMBP的研究现状和最新进展.     

应用磷酸二酯酶定量测定植物钙调素

钙调素(Calmodulin,简称CaM)研究工作中一个重要手段是其定量测定。目前国内外CaM定量方法主要有3类:酶法(如PDE酶、NAD激酶等);免疫学方法(如放射免疫法等)和聚丙烯酰胺凝胶电泳法。其中环核苷酸磷酸二酯酶(Phosphodiesterase简称PDE酶)法因为可以测定活性态CaM,无需特殊设备或试剂,灵敏度虽低于放免法,但高于电泳法,是目前最为普遍应用的CaM定量方法。下面介绍我们在定量测定植物CaM中的做法与体会。     

植物及动物钙调素抗体免疫反应特性的比较研究

用酶联免疫吸附测定和胶体金免疫电镜定位技术对三种钙调素抗体与植物和动物钙调素的免疫反应特性进行了比较研究。结果表明,在与小麦钙调素的相对亲和力中,抗小麦钙调素抗体大于抗 DNP 修饰猪脑钙调素抗体,抗牛脑钙调素抗体则很弱。抗小麦钙调素抗体与小麦钙调素的 K_D(解离常数)值为2.50×10~(-9)mol/L;抗 DNP 修饰猪脑钙调素抗体与小麦钙调素的 K_D 值为2.82×10~(-8)mol/L,而它与牛脑钙调素的 K_D 值为1.90×10~(-)mol/L。定位玉米根尖细胞钙调素,抗小麦钙调素抗体比抗 DNP 修饰猪脑钙调素抗体有更高的标记密度.定量小麦钙调素,抗小麦钙调素抗体比抗 DNP 修饰猪脑钙调素抗体有较高的检测灵敏度。     

钙调素及钙调素相关蛋白在植物细胞中的研究进展

植物对一系列生物和非生物刺激所产生的反应都与细胞内Ca2+信号转导有关,而钙调素、钙调素相关蛋白则是Ca2+信号转导的下游靶蛋白。该文介绍了钙调素的结构及其在植物细胞中的分布,钙调素及钙调素相关蛋白在植物细胞中的表达等方面的最近研究进展。     

生物素标记钙调素用于植物钙调素结合蛋白的检测

用生物素标了己了花椰菜ＣａＭ。生物素标记的ＣａＭ具有与天然ＣａＭ相似的Ｃａ２＋依赖电泳特性,可激活ＣａＭ依赖性磷酸二酯酶,能够检测出５０ｎｇ的磷酸二酯酶。利用它建立了检测植物ＣａＭ结合蛋白的生物素－覆盖法（Ｂｉｏｔｉｎ－ｏｖｅｒｌａｙ）并证实酶标亲和素可与胡萝卜愈伤组织内６４ｋＤ蛋白质非特异结合,因此将此法运用于植物材料时必需设置酶标亲和素处理的对照。用生物素－覆盖法检测胡萝卜愈伤组织形成过程中的ＣａＭ结合蛋白时可检出２,４－Ｄ诱导的ＣａＭ结合蛋白。     

植物钙调素的纯化和鉴定

钙调素(Calmodulin,CaM)是一种酸性、小分子量的单肽链调节蛋白。它是Cheung1967年首先在牛脑中发现的。Anderson和Cormier于1978年证实豌豆中也存在CaM。现在已知CaM广泛存在于真核生物中,也可能存在于原核生物中。CaM在多种植物生     

人钙调素在大肠杆菌中的表达、纯化及其活性研究

利用基因重组技术,将经PCR扩增获得的人钙调素基因(hCaMcDNA)插入质粒pBV220,构建重组表达载体hCaM/pBV220,用酶切、DNA测序、PCR扩增鉴定阳性克隆.阳性重组子在大肠杆菌DH5α中经温度诱导可高效表达CaM蛋白,经15%SDS-PAGE分析,可观察到一与CaM分子量相符(约17kD)的诱导表达条带,其表达量占菌体蛋白总量20%,并主要以可溶性形式表达.Westernblot结果证实,17kD的表达条带可与标准鼠抗人CaM单克隆抗体起特异反应.用Pheny1-SepharoseCL-4B疏水亲和层析法纯化重组菌超声上清表达产物,每1L菌液可获CaM纯品3～4mg.重组人CaM(rhCaM)与牛脑CaM的氨基酸组成基本一致.生物活性测定结果提示,rhCaM具有激活NAD激酶的活性,其激活程度与标准人脑CaM几乎一致.     

35S标记重组植物钙调素的制备方法

利用35S标记的氨基酸混合物喂养工程菌,成功地制备了35S标记的拟南芥钙调素亚型2(35S-ACaM2),对其纯度、放射活度、电泳行为及其灵敏性等进行了检测.结果表明从工程菌中制备的35S-ACaM2纯度高、放射活度高、Ca2+与EGTA存在时的电泳行为与未标记的ACaM2相同,可作为一种高灵敏性的探针用于检测钙调素结合蛋白.     

植物胞外钙调素与信号转导

植物体需要构建复杂的信号转导体系以调节自身的生长发育过程并适应外界环境的变化,这种功能的实现需要胞内和胞外诸多信号分子的参与,胞外钙调素的发现使人们开始相信植物细胞外多肽信使的存在。胞外钙调素的生物学功能极其广泛,几乎涉及到植物生长发育的各个阶段,其信号转导途径是目前研究得最多也是最为清楚的方面,异三聚体G蛋白、磷脂酶C(PLC)-肌醇三磷酸(IP3)-肌醇三磷酸受体(IP3R)信号通路、活性氧和Ca2 通道之间直接或间接的相互作用是胞外钙调素信号转导的核心。     

Single-Channel Resolution of the Interaction between C-Terminal CaV1.3 Isoforms and Calmodulin

Voltage-dependent calcium (Ca_V) 1.3 channels are involved in the control of cellular excitability and pacemaking in neuronal, cardiac, and sensory cells. Various proteins interact with the alternatively spliced channel C-terminus regulating gating of Ca_V1.3 channels. Binding of a regulatory calcium-binding protein calmodulin (CaM) to the proximal C-terminus leads to the boosting of channel activity and promotes calcium-dependent inactivation (CDI). The C-terminal modulator domain (CTM) of Ca_V1.3 channels can interfere with the CaM binding, thereby inhibiting channel activity and CDI. Here, we compared single-channel gating behavior of two natural Ca_V1.3 splice isoforms: the long Ca_V1.3₄₂ with the full-length CTM and the short Ca_V1.3_42A with the C-terminus truncated before the CTM. We found that CaM regulation of Ca_V1.3 channels is dynamic on a minute timescale. We observed that at equilibrium, single Ca_V1.3₄₂ channels occasionally switched from low to high open probability, which perhaps reflects occasional binding of CaM despite the presence of CTM. Similarly, when the amount of the available CaM in the cell was reduced, the short Ca_V1.3_42A isoform showed patterns of the low channel activity. CDI also underwent periodic changes with corresponding kinetics in both isoforms. Our results suggest that the competition between CTM and CaM is influenced by calcium, allowing further fine-tuning of Ca_V1.3 channel activity for particular cellular needs.     

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca²⁺-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca²⁺-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca²⁺-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca²⁺/CaM. Ca²⁺/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca²⁺/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca²⁺ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca²⁺/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.     

Calmodulin

Summary Ca²⁺ as an important cellular regulator has long been recognized. Calmodulin is unique among several proteins considered to be Ca²⁺ receptors in its ubiquitous distribution in eukaryotic cells and in its multiple effects through interaction with different enzymes and proteins. Apparently, calmodulin is the major Ca²⁺ receptor in most of these cells and most of metabolic active Ca²⁺ exists as a Ca²⁺-calmodulin complex.The importance of calmodulin as a Ca²⁺ mediator is also indicated by its role as the Ca²⁺-sensor in the regulation of Ca²⁺ pump which effectively maintains a low steady level of intracellular free Ca²⁺. The participation of calmodulin in the regulation of intracellular Ca²⁺ level suggests the desire for the cell to maintain adequate steady levels of metabolic active Ca²⁺. A low calmodulin concentration may in effect slow down the Ca²⁺ pump allowing a higher concentration of intracellular free Ca²⁺, but may also require higher Ca²⁺ threshold for Cat+ effects. A prominent difference in calmodulin contents of different eukaryotic cells has been noted and this difference may reflect the difference in the extents and the types of Ca²⁺-mediated reactions that operate in the cells. It is also possible that calmodulin concentration may fluctuate in response to different metabolic conditions. The evident for such possibility has been provided by the observations that cAMP-dependent protein kinase and ATP together with cAMP or neurotransmitters that stimulate cAMP synthesis cause the release of calmodulin from synaptic membranes (139, 140). However, the cytosolic calmodulin increased as the result of its release from the membranes is unlikely to be sufficient for eliciting calmodulin-mediated Ca²⁺ effects without a concomitant significant increase of intracellular Ca²⁺. The calmodulin release, in effect, may decrease the Ca²⁺ threshold of these effects.The manifestation of calmodulin-mediated Ca²⁺ effects in a particular type of cells appears determined mainly by the calmodulin-regulated enzymes existing in the cells. Within the same cells, however, the particular species of Ca²⁺-calmodulin complex serving as the active calmodulin, the affinity of the enzyme for the active calmodulin and the localization of the enzyme in the cells may determine the circumstance under which particular reactions are expressed.During the past years, substantial progress has been made in understanding calmodulin in terms of primary structure and molecular properties and in discovering many Ca²⁺-dependent, calmodulin-regulated enzymes and cellular activities. Our understanding of calmodulin and its relation to the wide range of Ca²⁺-dependent enzymes and activities has provided a framework for comprehending Ca²⁺ functions in the cells at the molecular level. Further works, however, are required to unravel fully the detailed mechanisms and properties that govern the calmodulin-enzyme interactions and to narrow further the gaps between Ca²⁺-elicited cellular expressions and the molecular events that lead to such expressions.     

Parvalbumin Isoforms in Zebrafish

By using an analysis of existing genomic information it is concluded that in zebrafish nine genes encode parvalbumin (PV). These genes possess introns that differ in size and show nucleotide variability but they contain the same number of exons, and for each corresponding exon, the number of nucleotides therein are identical in all the paralogs. This rule also applies to the multiple PV genes of other species e.g. mammals. Each of these genes displays, however, characteristic 5′ and 3′ UTRs which appear highly conserved between closely related species (so that orthologs among these species can be readily identified) but which show larger numbers of mutations between species that are more distant in evolution. A tree is presented which suggests that the traditional classification of PVs as alpha or beta (based mainly on charge of the protein molecule) is not sustainable. Numbers 1–9 are assigned to the various isoforms to facilitate their identification in future studies. A bifurcation of isoforms into 1 and 4; 2 and 3; 6 and 7; 8 and 9 appears to have occurred simultaneously in more recent time, i.e. perhaps approximately 60 mys ago when primates and rodents branched.     

Calmodulin in mammalian nerve

Calmodulin, a calcium-dependent regulatory protein has been isolated from mammalian nerve. The protein has similarities to the calcium-binding protein earlier shown to be transported at a fast rate in the nerve fibers. The implication is that calmodulin, which has been shown to be involved in various key cellular processes, may have a relation to axoplasmic transport.     

Calmodulin pharmacology

Calmodulin (CaM) is a major intracellular receptor for Ca²⁺. CaM is thus a crucial receptor to consider in pharmacological modification of cellular activity. Potential mechanisms by which drugs may modify CaM effectiveness are considered in the context of its interaction with Ca²⁺ and in turn with its various effectors. Some examples of established drug mechanisms are considered. A wide range of chemical compounds representing diverse pharmacological classes are anti-CaM under some conditions. No simple relationships have been established between molecular level events and therapeutic applicability of anti-CaM compounds.