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.     

拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用

动植物系统研究表明,钙调素不仅在结合钙离子时调节多种靶酶或靶蛋白的活性,而且没有钙离子结合时,还可以通过结合钙不依赖的钙调素结合蛋白,发挥多种生物学作用．然而,目前却没有体内分析钙调素与钙不依赖钙调素结合蛋白相互作用的方法．首先,采用定点突变的方式,得到了拟南芥钙调素亚型2的多个突变基因mCaM2,随后,大肠杆菌重组表达突变蛋白的电泳迁移率及⁴⁵Ca²⁺覆盖分析表明,得到了编码失去钙结合能力的钙调素的突变基因mCaM2₁₂₃₄, mCaM2₁₂₃₄突变钙调素中所有4个钙结合EF-hand结构域中的关键氨基酸谷氨酸均突变为谷氨酰胺．在酵母双杂交体系中,作为诱饵蛋白的突变钙调素mCaM2₁₂₃₄与我们前期体外方法报道的钙不依赖性钙调素结合蛋白AtIQD26存在相互作用．这将为钙不依赖性钙调素结合蛋白提供有用的体内研究工具,有利于我们全面认识钙-钙调素-钙调素结合蛋白信号途径．     

钙依赖的磷脂结合蛋白——钙结合蛋白中的一个新家族

钙依赖的磷脂结合蛋白是70年代末发现的一类新的钙结合蛋白,它们不同于钙调素等具有“EF”手结构的钙结合蛋白,其特点是它们与Ca²⁺结合后可以进一步与膜磷脂结合。这类蛋白质广泛存在于动物细胞,常常与质膜或内膜系统相联系。免疫化学证据和对其氨基酸顺序、cDNA序列分析表明,这是钙结合蛋白中一个包括多个成员、结构与功能相关的新家族。     

钙/钙调素信号途径在胁迫中的作用研究进展

钙调素普遍存在于真核生物细胞中,是多种生物学作用的信号组分.钙/钙调素信号途径由钙离子,钙调素以及下游的靶蛋白组成,通过与靶蛋白作用而传递信号并且发挥生物学功能.本文主要对于旱,盐,冷以及热胁迫下钙调素结合蛋白的作用进行综述,并对相关研究领域的未来研究方向进行了展望.     

钙／钙调素依赖性蛋白激酶对17．7kD和6kD胰腺蛋白的磷酸化

本文报导了胰腺提取物中两种可被钙／钙调素依赖性蛋白激酶磷酸化的热稳定蛋白。ＳＤＳ－ＰＡＧＥ测定其表观分子量分别为１７．７ｋＤ和６ｋＤ。经钙／钙调素依赖性蛋白激酶磷酸化后,其最大磷酸参入量为８．８μｍｏｌ／ｇ蛋白。同时磷酸化作用导致１７．７ｋＤ蛋白在ＳＤＳ－ＰＡＧＥ中迁移率发生变化。本文还进一步分析了各种阳离子对磷酸化的影响,并对此两种蛋白可能具有的生理功能进行了初步探讨。     

钙调素结合蛋白参与调控植物逆境胁迫的研究进展

Ca²⁺是植物体内重要的第二信使,当植物受到各种环境刺激时,细胞内的Ca²⁺浓度瞬间产生变化,并被Ca²⁺信号效应器识别,通过与下游的靶蛋白结合并调节其活性,参与调控植物各种生理活动。钙调素结合蛋白以依赖Ca²⁺或不依赖Ca²⁺的方式结合钙调素。对目前已经鉴定的植物钙调素结合蛋白结构特点进行了综述,并着重介绍了钙调素结合蛋白是如何参与调节植物对生物胁迫和非生物胁迫的反应,为提高作物抗病抗逆能力研究提供理论基础。     

小麦黄化胚芽鞘的细胞壁钙调素和钙调素结合蛋白

小麦黄化胚芽鞘经苯基琼脂糖亲和层析提取和纯化,其细胞壁CaM在有钙和缺钙时SDS电泳呈现不同的迁移率;依赖Ca~(2 )与苯基疏水结合;在紫外吸收光谱上具有五个特征峰;对PDE的激活剂量反应曲线和从非活性状态向活性状态转变时所需的Ca~(2 )浓度均和胞内CaM相同,说明细胞壁CaM和胞内CaM具有相同的基本理化特性。采用CaM琼脂糖亲和层析,发现在小麦细胞壁中存在CaM结合蛋白,其中以分子量为40.7 kD的 CaM结合多肽为主。细胞壁CaM结合蛋白不具有过氧化物酶、ATP酶或酸性磷酸酯酶的活性。     

钙-钙调素与小麦苗中热激蛋白的诱导

在 34℃热激条件下 ,种子经钙预处理的小麦苗中的钙调素含量随着热激时间的延长而增加 ,热激 90min时达最大值 ,而种子用钙离子螯合剂EGTA预处理的小麦苗中钙调素含量无明显增加。种子用EGTA及钙调素拮抗剂CPZ和TFP预处理的小麦幼苗在 34℃热激时 ,热激蛋白的合成量减少。 4d的小麦幼苗在34℃或 37℃热激条件下 ,能诱导耐热性的获得 ,分别用EGTA、钙离子通道阻断剂易博定、钙调素拮抗剂TFP或CPZ预处理种子后 ,所得幼苗热诱导的耐热性的提高程度有所下降     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Calmodulin content,Ca2+-dependent calmodulin binding proteins,and testis growth: Identification of Ca2+-dependent calmodulin binding proteins in primary spermatocytes

In contrast with the transient pre-replicative increase in calmodulin (CaM) level observed in proliferative activated cells, postnatal development of rat testis was paralleled by 3 specific rises in CaM. The first one occurred between 5 and 10 days, coincident with the appearance and proliferation start of spermatogonia and Sertoli cells. Meiosis accomplishment and spermatid differentiation were paralleled by 2 additional rises, at 24 and 32 days, respectively. The plateau phase of testis growth was coincident with the appearance of maturating spermatids and spermatozoa in the germinal epithelium, and with a decrease in CaM content. Testicular DNA:g wet tissue ratio reached the highest level in 15-day-old rats and gradually decreased up to 35 days, when a constant level was reached. A similar level of Ca²⁺-CaMBPs was observed in 5- and 20-day-old rat testis. Although all subcellular fractions showed the ability to bind CaM in a Ca²⁺-dependent manner, CaM was mainly recovered in the nuclear and soluble fractions of adult and immature rat testis. Several Ca²⁺-CaMBPs with an apparent M_r of 82, 75, 64, 19, and 14 kD were purified by affinity chromatography from pachytene primary spermatocyte nuclear matrix. Ca²⁺-CaMBPs showing an M_r of 120, 78, 72, and 66 kD were also purified from the supernatant obtained after DNA and RNA hydrolysis of meiotic nuclei. Major cytosolic Ca²⁺-CaMBPs of primary spermatocytes showed an M_r of 120, 84, 44, and 39 kD. The functions that these Ca²⁺-CaMBPs might have during the first meiotic prophase is discussed. Mol. Reprod. Dev. 48:127–136, 1997. © 1997 Wiley-Liss, Inc.     

43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca²⁺泵(Ca²⁺-ATPase)是调节细胞内Ca²⁺浓度的重要蛋白质之一. Ca²⁺泵在转运Ca²⁺的过程中经历一系列构象变化. 其中,E1状态为外向的Ca²⁺高亲和状态,E2状态则为内向的Ca²⁺低亲和状态. 目前,骨骼肌内质网Ca²⁺泵转运Ca²⁺过程中的几个中间状态,包括E1-2Ca²⁺,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析. 介绍这几种状态的晶体结构,并分析Ca²⁺泵在执行功能过程中结构与功能的关系.