Inhibition of photosynthetic oxygen evolution by calmodulin-type inhibitors and other calcium-antagonists

Several studies have recently implicated a role for Ca2+ in photosynthetic oxygen evolution (9-11). Our previous study indicated that Ca2+ was likely acting at the level of the Cl- cofactor requirement in photosystem II (9). We now demonstrate, through the use of calmodulin-type inhibitors ( calmidazolium and trifluoperazine) and metal Ca2+-antagonists (e.g., Tb+3 and La+3), the function of Ca2+ on the oxidizing side of photosystem II. In addition, the peroxide (H2O2) electron donation site was differentiated from the electron donation site of NH2OH, Mn2+ and diphenyl carbazide in the photosystem II complex.     

丹参酮lI-A磺酸钠及莨菪等药物对红细胞膜稳定性及钙调节蛋白(CaM)功能的影响

 为了进一步证实某些药物对红细胞膜稳定(即抗溶血)作用及抑制CaM功能与药理作用之间的相关性,本文选用丹参酮Ⅱ-A磺酸钠,戊脉安,奎尼丁和莨菪类药物,测定它们对大鼠红细胞溶血及CaM功能的影响。结果表明丹参酮Ⅱ-A磺酸钠,戊脉安,奎尼丁对红细胞溶血抑制率分别为100％,71％,32％,对CaM功能的抑制分别为71.8％,63.4％,46.4％。莨菪类药物有促溶血作用,对CaM功能的抑制也极弱。我们还发现以上药物对膜上Ca~(2+)+Mg~(2+)-ATP酶的直接作用亦有类似的规律。这种相关性可能与膜表面CaM与钙离子,靶酶及药物结合区的疏水性及药物本身的相对疏水性有关。     

家兔钙调节蛋自-红细胞膜的制备

 本文介绍用等渗咪唑缓冲液溶血,并用低温高速或常速离心机制备出一种带钙调节蛋白(简称CaM)的红细胞膜。它具有与膜稳定结合的CaM,在钙离子存在下可以激活膜上的靶酶——Ca~(2+)+Mg~(2+)-ATP酶活性,为研究CaM功能及有关药物机制提供一种简便而理想的材料。     

牛脑Ca~(2+)/CaM依赖性蛋白激酶Ⅱ的纯化和鉴定

通过一系列层析法,首次从牛脑纯化得到胶凝电泳匀一的Ca~(2+)/CaM PKⅡ。凝胶过滤法测定全酶分子量为550kD,SDS-PAGE法测定亚基分子量为55kD,推测牛脑Ca~(2+)/CaM PK Ⅱ由十个相同的亚基组成。该酶活性绝对依赖于Ca~(2+)和CaM,以63kD PDE同工酶为底物,其AC_(50)分别为0.85μmol/L和0.18μmol/L;以酪蛋白为底物,其AC_(50)分别为0.22μmol/L和0.06μmol/L。牛脑Ca~(2+)/CaM PK Ⅱ旣能催化63kD PDE同工酶等多种蛋白或酶磷酸化,又能进行自身磷酸化。该酶催化63kD PDE同工酶最大磷酸参入量为1mol/mol亚基。磷酸化型63kD PDE同工酶的Ca~(2+)的AC_(50)高于非磷酸化型。     

红光对绿豆下胚轴线粒体Ca~(2+)积累、ATPase及NAD激酶活性的影响

绿豆下胚轴切段经红光处理后10min,其线粒体的Ca~(2+)积累下降15％,Ca~(2+)-ATPase 及 Mg~(2+)-ATPase活性也分别下降29％和10％,切段CaM含量增加近1倍。Ca~(2+)存在时,红光能促进线粒体NAD 激酶活性。说明Ca~(2+)-ATPase及一部分Mg~(2+)-ATPase可作为钙泵控制Ca~(2+)进入线粒体。     

钙调素拮抗剂对人肺癌细胞增殖抑制及与cAMP信号系统相关性之初探

对钙调素（ＣａＭ）拮抗剂—三氟拉嗪（ｔｒｉｆｌｕｏｐｅｒａｚｉｎｅ,ＴＦＰ）在人肺癌细胞ＰＬＡ８０１的增殖抑制中的作用和ＣａＭ与ｃＡＭＰ信号系统水平的变化进行了研究．用５、１０、１５和２０μｍｏｌ／ＬＴＦＰ处理人肺癌细胞时观察到ＴＦＰ在抑制细胞内ＣａＭ活性的同时,抑制了细胞的增殖．药物处理的细胞在软琼脂中形成的集落数减少且明显小于对照组细胞．使用流式细胞光度术分析细胞周期的结果表明：１０μｍｏｌ／ＬＴＦＰ处理抑制了Ｇ１期细胞向Ｓ期的转移．当用１０μｍｏｌ／ＬＴＦＰ作用细胞５ｍｉｎ时,细胞内ｃＡＭＰ水平达到正常水平的１．８倍,直到３ｈ仍明显高于正常水平．同时,ｃＡＭＰ依赖的ＰＫＡ的活性在加药后１５ｍｉｎ上升到正常水平的２．８倍,直到加药３ｈ．活性仍保持较高水平,结果表明：钙调素功能的抑制,提高了ＰＬＡ－８０１细胞内ｃＡＭＰ系统的水平,Ｃａ２＋－ＣａＭ和ｃＡＭＰ－ＰＫＡ两个信号系统的协调作用,抑制了细胞的增殖     

Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by β-arrestin-1 and requires an essential serine residue in the receptor C-tail

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys³⁵⁹-Ser³⁶⁰-Arg³⁶¹. Moreover, point mutation of Ser³⁶⁰ to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of β-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser³⁶⁰ being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with β-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.     

细胞外钙调素对烟草悬浮培养细胞蛋白质磷酸化作用的影响

用液体闪烁计数法研究了细胞外钙调素对烟草悬浮培养细胞质蛋白质磷酸化的作用。结果表明烟草细胞细胞质蛋白质磷酸化活性在细胞培养过程中逐渐增加,达到最高峰后又开始下降。在细胞质蛋白质磷酸化强度高峰时,加入抗CaM血清后,细胞质蛋白质磷酸化活性受到了部分抑制。加抗CaM血清后再补加CaM能够部分解除抗CaM血清对细胞质部分与细胞核部分蛋白质磷酸化的抑制作用。外加纯化钙调素可以引起烟草悬浮培养细胞细胞质蛋白质磷酸化的活性增强,并且这种增强作用具有时间（高峰为70min）与剂量（最适为CaM10-7mmol/L）依赖性。CaM引起的细胞质蛋白质磷酸化变化与红光所引起的细胞质蛋白质磷酸化变化在时间进程上是不相同的。     

Crystal-structure and biochemical characterization of recombinant human calcyphosine delineates a novel EF-hand-containing protein family

Calcyphosine is an EF-hand protein involved in both Ca^2 +-phosphatidylinositol and cyclic AMP signal cascades, as well as in other cellular functions. The crystal structure of Ca^2 +-loaded calcyphosine was determined up to 2.65 Å resolution and reveals a protein containing two pairs of Ca^2 +-binding EF-hand motifs. Calcyphosine shares a highly similar overall topology with calmodulin. However, there are striking differences between EF-hand 4, both N-terminal and C-terminal regions, and interdomain linkers. The C-terminal domain of calcyphosine possesses a large hydrophobic pocket in the presence of calcium ions that might be implicated in ligand binding, while its N-terminal hydrophobic pocket is almost shielded by an additional terminal helix. Calcyphosine is largely monomeric, regardless of the presence of Ca^2 +. Differences in structure, oligomeric state in the presence and in the absence of Ca^2 +, a highly conserved sequence with low similarity to other proteins, and phylogeny define a new EF-hand-containing family of calcyphosine proteins that extends from arthropods to humans.     

Mastoparan/Mastoparan X altered binding behavior of La3+ to calmodulin in ternary complexes

Ca(2+) binds to calmodulin (CaM) and triggers the interaction of CaM with its target proteins; CaM binding proteins (CaMBPs) can also regulate the metal binding to CaM. In the present paper, La(3+) binding to CaM was studied in the presence of the CaM binding peptides, Mastoparan (Mas) and Mas X, using ultrafiltration and titration of fluorescence. Ca(2+) binding was used as an analog to understand La(3+) binding in intact CaM and isolated N/C-terminal CaM domain of metal-CaM binary system and metal-CaM-CaMBPs ternary system. Mas/Mas X increased binding affinity of La(3+) to CaM by 0.5 approximately 3 orders magnitude. The metal ions binding affinity to the C-terminal or the N-terminal CaM domain suggested that in the first phase of binding process both Ca(2+) and La(3+) bind to C-terminal of CaM in the presence of Mas/Mas X. In the presence of CaM binding peptides, La(3+) binding preference was substantially altered from the metal-CaM binary system where La(3+) slightly preferred binding to the N-terminal sites of CaM. Our results will be helpful in understanding La(3+) interactions with CaM in the biological systems.