PICK1蛋白C端酸性区域对其功能的调节

蛋白激酶C相互作用蛋白1(protein interacting with Ckinase1,PICK1)是调节AMPA(alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)受体在细胞膜上的数量与分布,引起LTP与LTD现象的重要蛋白.本文利用基因克隆、荧光光谱以及免疫分析等方法,分析了PICK1蛋白C末端酸性区对BAR结构域与膜脂结合能力以及PICK1分子内BAR(Bin/amphiphysin/RVS)结构域与PDZ结构域相互作用的影响,研究了钙离子结合C末端酸性区后对上述相互作用的调节.结果显示,C末端酸性区的存在使BAR结构域与膜脂的结合能力减弱大约10倍,但PICK1分子内的BAR与PDZ结构域的相互作用与不含C末端的酸性区相比增强了大约4倍.另一方面,C末端酸性区的存在,伴随钙离子浓度的提高,有助于增强BAR与膜脂的结合,却削弱了PDZ和BAR结构域的作用.当钙离子浓度增加到500μmol/L时,BARC的脂质结合能力以及和PDZ的亲和力与不含酸性区相当.     

植物细胞Ca2+荧光蛋白指示剂研究进展

Ca2+作为第二信使参与了植物生长和发育过程的调控,不同生物和非生物胁迫信号均可诱导胞内Ca2+变化.对Ca2+在信号转导作用中的认识主要来自于细胞内Ca2+浓度测定.水母发光蛋白和基于荧光蛋白的Ca2+荧光指示剂作为检测细胞Ca2+信号的手段是近年发展起来的新方法.本文综述了水母发光蛋白和基于荧光蛋白的Ca2+荧光指示剂的发展、测量原理、优点与不足及其在细胞Ca2+信号转导中的应用研究进展.     

植物Mg2+转运蛋白与相关基因及其功能的研究进展

镁离子是植物细胞中最丰富的二价阳离子.在植物的生长发育中起着重要的作用.对Mg2+独特的几何和化学性质,Mg2对植物细胞体内中动态平衡的影响,植物中Mg2+转运相关蛋白与相关基因及其功能的研究进展进行了综述.     

蛋白激酶Cα相互作用蛋白的结构与功能

蛋白激酶Cα相互作用蛋白（protein interacting with Cα kinase,PICK1）是蛋白激酶Cox（protein kinase Cα,PKCα）的靶蛋白之一,也是在PKCα和突触后膜受体蛋白间起重要作用的衔接蛋白。PICK1分别由PDZ结构域、BAR结构域以及卷曲螺旋区和酸性氨基酸区组成。PICK1中的PDZ结构域和受体蛋白、转运蛋白、衔接蛋白的相互作用报道较多,BAR结构域则与支架蛋白、质膜等相互作用。PICK1在突触可塑性、神经递质传递、外周神经感觉、细胞生长和黏连等方面发挥重要作用。本文对PICK1的结构和功能进行综述。     

植物Mg转运蛋白与相关基因及其功能的研究进展

镁离子是植物细胞中最丰富的二价阳离子,在植物的生长发育中起着重要的作用．对Mg^2＋独特的几何和化学性质,Mg^2＋对植物细胞体内中动态平衡的影响,植物中Mg^2＋转运相关蛋白与相关基因及其功能的研究进展进行了综述．     

端粒蛋白TRF1与肌动蛋白结合蛋白PFN2相互作用的鉴定

目的:鉴定端粒蛋白TRF1和肌动蛋白结合蛋白PFN2是否存在相互作用,并且两者的相互作用是否与端粒在细胞核周的锚定有关。方法:将TRF1构建到myc标签载体,PFN2构建到GST标签载体,采用GST-pull down技术,验证两者是否存在相互作用;同时将TRF1构建到EGFP标签的绿色荧光载体,PFN2构建到RED标签的红色荧光载体,两者共转入细胞,利用荧光显微镜观察两者在细胞中的共定位情况。结果:GST-pull down证明TRF1与PFN2存在直接相互作用,两者在细胞中可以共定位。结论:TRF1与PFN2存在相互作用,且这种相互作用发生在细胞核周。     

蛋白激酶Ca相互作用蛋白的结构与功能

蛋白激酶Cα相互作用蛋白(proteininteractingwithCαkinase,PICK1)是蛋白激酶Cα(proteinkinaseCα,PKCα)的靶蛋白之一,也是在PKCα和突触后膜受体蛋白间起重要作用的衔接蛋白。PICK1分别由PDZ结构域、BAR结构域以及卷曲螺旋区和酸性氨基酸区组成。PICK1中的PDZ结构域和受体蛋白、转运蛋白、衔接蛋白的相互作用报道较多,BAR结构域则与支架蛋白、质膜等相互作用。PICK1在突触可塑性、神经递质传递、外周神经感觉、细胞生长和黏连等方面发挥重要作用。本文对PICK1的结构和功能进行综述。     

低温胁迫下拟南芥CBF1 超表达突变体胞质中Ca2+ 浓度的变化

低温严重影响植物的生长, 低温刺激可引起植物细胞中Ca²⁺浓度迅速升高。以拟南芥(Arabidopsis thaliana) CBF1 超表达突变体为材料, 研究了低温处理时CBF1基因的表达情况及胞质Ca²⁺的浓度变化。结果表明, CBF1本身可受低温诱导。同时将水母发光蛋白基因转入该拟南芥突变体中并检测Ca²⁺的浓度变化, 发现低温刺激时突变体细胞质中Ca²⁺的浓度变化幅度明显高于野生型, 但液泡的胞质面两侧Ca²⁺的浓度变化相似。用EGTA和LaCl₃处理拟南芥后, 胞质Ca²⁺的浓度升高被抑制, 并且CBF1突变体及对照胞质中的Ca²⁺浓度下降到同一水平。上述结果表明, Ca²⁺参与了CBF1应答低温信号的转导过程, 并且CBF1超表达突变体可能是通过提高胞质Ca²⁺浓度来提高植物的抗低温胁迫能力。     

重组人睾丸精子结合蛋白对人精子蛋白激酶A及蛋白激酶C活性的影响

为探讨人睾丸精子结合蛋白(human testis sperm binding protein,hTSBP)促进人精子获能的分子机制,检测了重组hTSBP对人精子蛋白激酶A(PICA)和蛋白激酶C(PKC)活性的影响.用重组载体pcDNA3.1/myc-His(-)B-tsbp转染真核细胞后,金属亲和层析纯化表达的重组蛋白质His6-TSBP;重组蛋白质处理健康成人精子后,分别提取精子胞浆及胞膜蛋白组分,发现重组蛋白质可以与精子胞膜结合;经放射自显影检测,发现0.1 mg/ml重组蛋白质可以提高人精子中PKA的活性,对精子膜PKC活性无显著影响.     

Z-DNA结合蛋白与Zα结构域

Z-DNA结合蛋白包括人、哺乳类和病毒中的ADAR1,DLM-1.E3L蛋白以及在鱼类中最新报道的PKR-like(PKZ),其共同点是都含有za结构域.本文简述了这几种蛋白质的结构与功能,并着重分析Za结构域与Z-DNA,Z-RNA、B-DNA 等核酸分子的识别机制和亲和性.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Effects of diet and temperature on Morimus funereus larval hemolymph cation concentrations

The effects of diet and different constant temperatures on hemolymph cation concentrations (Na⁺, K⁺, Mg²⁺, Ca²⁺) have been studied in Morimus funereus larvae collected from natural habitat, fed natural (oak or beech bark) or artificial diet, as well as in larvae reared from hatching on an artificial diet. In the hemolymph of larvae maintained under natural conditions Mg²⁺ was dominant, whereas Na⁺ concentration was very low. In their natural diets concentrations of Na⁺ and K⁺ were very low, while those of Ca²⁺ and Mg²⁺ were high. In larvae continuously reared on an artificial diet, hemolymph Mg²⁺ concentration was significantly decreased and Na⁺ concentration increased more than fourfold compared to the results obtained in oak-fed larvae. Na⁺ and K⁺ are the dominant cations in the artificial diet. The concentrations of K⁺ and Ca²⁺ in the hemolymph of larvae fed natural or artificial diet are nearly identical, suggesting the existence of an internal regulatory mechanism in this insect for these cations. The hemolymph cation concentrations of M. funereus larvae are predominantly dependent upon the diet consumed, much less upon the environmental temperatures. The most stable concentrations of cations were observed in larvae continuously fed an artificial diet and exposed to different constant temperatures. There was much less stability in the hemolymph cation concentration in oak larvae fed either natural or artificial food after their transfer to constant temperatures. With respect to the response to the external factors studied, the most sensitive are the Na⁺ concentrations, the most stable seems to be K⁺. © 1992 Wiley-Liss, Inc.     

Magnesium-dependent induction of phagocytosis-associated chemiluminescence of adherent human polymorphonuclear leukocytes by non-opsonized zymosan

A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) to non-opsonized zymosan was observed under a deficiency of extracellular Mg²⁺. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg²⁺-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca²⁺ did not compensate for the Mg²⁺ deficit. In the presence of Mg²⁺ only a slight Ca²⁺-dependent reduction of NL was obtained, but Ca²⁺ seemed to selectively promote LDL. This may indicate a positive influence of Ca²⁺ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg²⁺ to differing extents, confirmed the important role of Mg²⁺ in PMNL-activation by non-opsonized zymosan.     

血管紧张素Ⅱ对大鼠心肌肌浆网Ca~(2+),Mg~(2+)-ATPase基因转录调节的影响

观察血管紧张素Ⅱ（ＡｎｇⅡ）对心肌肌浆网Ｃａ２＋,Ｍｇ２＋－ＡＴＰａｓｅ基因（ＳＥＲＣＡ２ａ）转录调节的影响,评价ＤＭＰ８１１对此效应的干预作用．６周龄雄性ＳＤ大鼠随机分为３组,每组６只．组１：生理盐水输注;组２：ＡｎｇⅡ输注＋ＤＭＰ８１１管饲（３ｍｇ·ｄ－１·ｋｇ－１）;组３：ＡｎｇⅡ输注（２００ｎｇ·ｍｉｎ－１·ｋｇ－１．１周后称其体重,取心脏并称重,提取心脏总ＲＮＡ后采用Ｎｏｒｔｈｅｒｎｂｌｏｔ的方法检测ＳＥＲ－ＣＡ２ａ的转录水平,采用ＲＴ－ＰＣＲ检测ＡｎｇⅡ１型受体（ＡＴ１）ｍＲＮＡ水平．实验后,组３心重（ＣＷ）、心重／体重（Ｃ／Ｂ）、ＡＴ１受体转录水平均高于组１（分别增加４．７±０．４％,４．９±０．９％和２４．７±３．５％;Ｐ＜０．０１）,而ＳＥＲＣＡ２ａ基因转录水平显著低于组１（降低２０．１±３．０％,Ｐ＜０．０１）,并且ＳＥＲＣＡ２ａｍＲＮＡ水平与ＡＴ１受体ｍＲＮＡ水平呈负相关（ｒ＝－０．７４,Ｐ＜０．０１）．ＡｎｇⅡ导致的上述改变能被ＤＭＰ８１１完全阻断．ＡｎｇⅡ通过其Ⅰ型受体的介导,诱导了ＳＥＲＣＡ２ａ的转录下调     

Stimulation of Ca efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate

We investigated the restoration of [Ca²⁺]_i in fura-2-loaded human platelets following discharge of internal Ca²⁺ stores in the absence of external Ca²⁺. After stimulation by thrombin [Ca²⁺]_i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca²⁺]_i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca²⁺]_i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca²⁺ efflux, perhaps via protein kinase C actions on a plasma membrane Ca²⁺ pump.     

The role of the Na+/Ca2+ exchangers in Ca2+ dynamics in ventricular myocytes

The role of the Na⁺/Ca²⁺ exchanger (NCX) as the main pathway for Ca²⁺ extrusion from ventricular myocytes is well established. However, both the role of the Ca²⁺ entry mode of NCX in regulating local Ca²⁺ dynamics and the role of the Ca²⁺ exit mode during the majority of the physiological action potential (AP) are subjects of controversy. The functional significance of NCXs location in T-tubules and potential co-localization with ryanodine receptors was examined using a local Ca²⁺ control model of low computational cost. Our simulations demonstrate that under physiological conditions local Ca²⁺ and Na⁺ gradients are critical in calculating the driving force for NCX and hence in predicting the effect of NCX on AP. Under physiological conditions when 60% of NCXs are located on T-tubules, NCX may be transiently inward within the first 100 ms of an AP and then transiently outward during the AP plateau phase. Thus, during an AP NCX current (I_NCX) has three reversal points rather than just one. This provides a resolution to experimental observations where Ca²⁺ entry via NCX during an AP is inconsistent with the time at which I_NCX is thought to become inward. A more complex than previously believed dynamic regulation of I_NCX during AP under physiological conditions allows us to interpret apparently contradictory experimental data in a consistent conceptual framework. Our modelling results support the claim that NCX regulates the local control of Ca²⁺ and provide a powerful tool for future investigations of the control of sarcoplasmic reticulum (SR) Ca²⁺ release under pathological conditions.     

Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647

Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. Ca2+-dependent conformational changes in recoverin are allosterically controlled by the covalently attached myristoyl group. The amino acid sequence of recoverin harbors a unique cysteine at position 38. The cysteine can be modified by the fluorescent dye Alexa647 using a maleimide-thiol coupling step. Introduction of Alexa647 into recoverin did not disturb the biological function of recoverin, as it can regulate rhodopsin kinase activity like unlabeled recoverin. Performance of the Ca2+-myristoyl switch of labeled recoverin was monitored by Ca2+-dependent association with immobilized lipids using surface plasmon resonance spectroscopy. When the Ca2+-concentration was varied, labeled myristoylated recoverin showed a 37%-change in fluorescence emission and a 34%-change in excitation intensity, emission and excitation maxima shifted by 6 and 18 nm, respectively. In contrast, labeled nonmyristoylated recoverin exhibited only minimal changes. Time-resolved fluorescence measurements showed biexponentiell fluorescence decay, in which the slower time constant of 2 ns was specifically influenced by Ca2+-induced conformational changes. A similar influence on the slower time constant was observed with the recoverin mutant RecE85Q that has a disabled EF-hand 2, but no such influence was detected with the mutant RecE121Q (EF-hand 3 is nonfunctional) that contains the myristoyl group in a clamped position. We conclude from our results that Alexa647 bound to cysteine 38 can monitor the conformational transition in recoverin that is under control of the myristoyl group.     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.