动物体液中的胞外钙调素结合蛋白

利用以生物素标记钙调素为探针的凝胶覆盖技术检测动物体液中胞外钙调素结合蛋白（ＣａＭＢＰ）。证明了动物中普遍存在胞外ＣａＭＢＰｓ，为胞外钙调素的作用机理提供了新线索。     

植物胞外CaM调节机理初探

利用ＮＡＤ激酶法和平衡透析法,作者研究了不同ｐＨ条件对Ｃａ＾２＋,钙调素（ＣａＭ）的结合能力及ＣａＭ激活ＮＡＤ激酶的影响。结果发现：Ｈ＾＋不仅能降低Ｃａ＾２＋与ＣａＭ的结合能力而且还能显著抑制ＣａＭ对ＮＡＤ激酶的激活。本实验证实了酸性条件下不利于ＣａＭ的活性状态的转变,即ｐＨ值低时激活ＣａＭ需要更多的Ｃａ＾２＋。此研究结果为植物胞外ＣａＭ的活性状态的转变,即ｐＨ值低时激活ＣａＭ需要更多的Ｃａ＾２＋     

蚯蚓钙结合蛋白的分离纯化及性质的研究

以赤子爱胜蚓为材料分离纯化了钙调素，并得到一种新的钙结合蛋白经ＳＤＳ－ＰＡＧＥ，ＰＡＧＥ和等电聚焦电泳鉴定，这两种蛋白均表现为一。ＣａＭ分子量为１８．９ｋＤ，ＮＣＢＰ为１６ｋＤ，等电点分别为３．６和４．３，两种蛋白具有不同的肽谱。     

白芷悬浮培养细胞外21KD钙调素结合蛋白的鉴定及纯化

利用生物素－ＣａＭ凝胶覆盖技术，在白芷悬浮培养细胞外检测一个分子量为２１ＫＤ的钙调素结合蛋白（ＣａＭｂｐ），并用ＳｅｐｈａｄｅｘＧ－１００凝胶过滤柱，ＣＭ－Ｓｅｐｈａｒｏｓｅ阳离子交换柱层析将其纯化。     

蚯蚓钙调素二级结构的研究及Ca^2＋,Tb^3＋的影响

运用ＦＴ－ＩＲ方法的分辨率增强及曲线拟合技术对水化膜中蚯蚓钙调素ＣａＭ的二级结构以及加入不同比例Ｃａ＾２＋、Ｔｂ＾３＋后ＣａＭ二级结构的变化进行了研究。结果表明，水化膜中ＣａＭ及含不同比例Ｃａ＾２＋或Ｔｂ＾３＋的蛋白复合物的α－ｈｅｌｉｘ的含量较晶体和溶液中为低，而β－ｓｈｅｅｔ和ｔｕｒｎ结构的含量相对较高。研究发现不同比例金属离子的加入，引起了ＣａＭ二级结构不同程度的变化，但各类结构在总量上观察     

重组人钙调素的表达及其McAb的制备与精子CaM定位

用基因重组技术构建人钙调素基因Ⅲ（ｈＣａＭⅢｃＤＮＡ）表达载体ｐＢＶ／ｈＣａＭⅢ,并在大肠杆菌ＤＨ５α中经热诱导获得可溶性ＣａＭ蛋白的高效表达．将纯化的重组ｈＣａＭ与异型双功能剂ＳＰＤＰ及鼠血清白蛋白（ＭＳＡ）交联,免疫Ｂａｌｂ／ｃ小鼠,用常规细胞融合技术制备单克隆抗体（ＭｃＡｂ）,得到３株稳定分泌单克隆抗体的杂交瘤细胞．间接ＥＬＩＳＡ和斑点免疫结果证实,三种单克隆抗体均与自制的ｒｈＣａＭ和ＣａＭ标准品起特异反应．用免疫组化法对精子中ＣａＭ定位,发现ＣａＭ主要分布于精子头颈部,不育组ＣａＭ＋精子率（（４５．０±７．５）％）显著低于生育组（（６８．５±１０．５）％）．     

植物钙调素的分离纯化及其特性研究

本文以烟草愈伤组织和花椰菜这花序为材料，分离纯化钙调素。所得ＣａＭ的纯度用ＳＤＳ－ＰＡＧＥ鉴定；生物活性用其对牛心肌环腺苷酸磷二酯酶（ＰＤＥ）的激活作用测定（ＣａＭ）的产量用酶联免疫吸附法（ＥＬＩＳＡ）测定。并比较了上Ｐｈｎｅｙｌ－ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ柱前对ＣａＭ溶液的不同处理。纯化的烟草愈伤组织和菜花ＣａＭ，具有牛脑ＣａＭ所特有的一些性质。它对牛心肌ＰＤＥ有明显激活作用，在含有Ｃａ＾２＋     

卵菌菌丝顶端钙调素的免疫组织化学定位

采用胞壁水解酶（Ｄｒｉｓｅｌａｓｅ２ｍｇ·Ｌ－１）和甲醇（－２０℃），分别对卵菌（Ｓａｐｒｏｌｅｇｎｉａｆｅｒａｘ）菌丝细胞壁及细胞膜进行通透，然后用免疫组织化学法对菌丝钙调素（ＣａＭ）的分布进行定位．结果表明ＣａＭ较密集地分布在菌丝顶端和在菌丝旁侧新形成的侧枝，亚顶端次之，基部菌丝几乎无特异性染色．由此提示，ＣａＭ可能参与卵菌菌丝顶端生长的调控．     

解脂假丝酵母胞外脂肪酶的提纯和性质

通过ＤＥＡＥ－ＣｅｌｌｕｌｏｓｅＤＥ－５２和ＤＥＡＥ－ＳｅＰｈａｄｅｘＡ－５０离子交换层析等提纯步骤，对解脂假丝酵母胞外脂肪酶进行了纯化，得到了层析纯样品，比活提高２７．４倍，得率１１％．该酶反应最适温度为４０℃；最适ＰＨ为７．５；等电点约在ＰＨ４．５～５．０之间；８℃以下存放，酶活力损失较少，３０℃以上存放，酶活力有明显损失．金属离子Ｓｒ２＋，Ｂａ２＋，Ｚｎ２＋和Ｍｇ２＋对该酶的激活作用为Ｓｒ２＋＞Ｂａ２＋＞Ｚｎ２＋＞Ｍｇ２＋．Ｃｕ２＋和Ｎａ＋对酶活性有抑制作用．ＮａＮ３对保持酶活性有重要作用．     

番茄碱对人细胞膜Ca^2＋—Mg^2＋ATPase活性影响的初步研究

本研究了番茄碱对人红细胞膜Ｃａ＾２＋－Ｍｇ＾２＋ＡＴＰａｓｅ活性的影响。实验结果表明，反应体系中ｔｏｍａｔｉｎｅ的浓度低于ｌｍｍｏｌ／Ｌ时，对不依赖钙调蛋白激活的Ｃａ＾２＋－Ｍｇ＾２＋ＡＴＰａｓｅ影响不大，浓度达ｌｍｍｏｌ／Ｌ时，该酶活性仍保持在９６％左右；而在此浓度范围内，ｔｏｍａｔｉｎｅ对依赖于ＣａＭ激活的Ｃａ＾２＋Ｍｇ＾２＋ＡＴＰａｓｅｅ有明显的抑制作用；其ＩＣ５０值为０．１４ｍｍｏｌ／Ｌ     

蚯蚓钙调素结合蛋白的研究

以赤子爱胜蚓(Eisenia Foetida)为材料,通过DEAE-Fast Flow离子交换层析、CaM-Sepharose亲和层析,分离纯化得到蚯蚓钙调素结合蛋白(CaMBPs)。纯化的CaMBPs对CaM激活的环核苷酸磷酸二酯酶活性有抑制作用,而且这种抑制作用可通过加入过量的CaM达到完全恢复。SDS-PAGE显示CaMBPs有3条明显主带,在EGTA存在时表现分子量分别为62, 49和30kD。紫外扫描测定含量分别为7.17%,7.31%和51.8%。用生物素-CaM覆盖法检测到3种CaM结合蛋白,与SDS-PAGE结果一致。酶活性测定实验表明在蚯蚓CaMBPs中有Ca²⁺-ATPase活性,但无NAD激酶活性。     

拟南芥悬浮培养细胞外钙结合蛋白的初步检测

钙离子通过与其结合的蛋白质在植物细胞信号转导中起着重要的作用,已知植物细胞外空间钙离子浓度高达毫摩尔级,而与钙结合的胞外蛋白有待进一步的分析研究.采用丙酮沉淀法分别制备了拟南芥悬浮培养细胞壁盐提蛋白及培养介质蛋白,制备的蛋白经SDS-PAGE电泳分离后,电转移至硝酸纤维素膜,45Ca2 覆盖结合,放射自显影,结果表明悬浮细胞外可能存在多条可与钙离子结合的蛋白条带,其分子量约为17,30,40,68 ku,推测这些蛋白可能参与胞外钙离子相关的生物学过程.     

葎草花粉主要过敏原的分离鉴定与纯化

采用液氮研磨法提取葎草花粉粗蛋白,凝胶电泳(SDS-PAGE)分离粗提液蛋白组分并测定各组分的相对分子质量,后经过离子交换层析法分离纯化几类主要的蛋白,再用westen blotting分别检测其与花粉过敏患者血清IgE结合情况,鉴定每种主要蛋白质致敏原的致敏性.分析了葎草花粉变应原成分及其变应原性、免疫原性,并鉴定主要过敏原的致敏性.SDS-PAGE凝胶电泳结果表明,广泛的分布于8～170 kDa之间有主带13条,次带10余条;westen blotting检测发现葎草花粉的致敏性较强,与花粉过敏患者血清IgE结合能力较高.离子交换层析出4种主要变应原蛋白,其相对分子质量分别为55,16,15和25 kDa,其中结合能力最强的是15 kDa处蛋白条带.葎草的主要过敏原为55,16,15和25 kDa蛋白质组分.     

Ca2+/calmodulin is critical for brassinosteroid biosynthesis and plant growth

Brassinosteroids are plant-specific steroid hormones that have an important role in coupling environmental factors, especially light, with plant growth and development. How the endogenous brassinosteroids change in response to environmental stimuli is largely unknown. Ca2+/calmodulin has an essential role in sensing and transducing environmental stimuli. Arabidopsis DWARF1 (DWF1) is responsible for an early step in brassinosteroid biosynthesis that converts 24-methylenecholesterol to campesterol. Here we show that DWF1 is a Ca2+/calmodulin-binding protein and this binding is critical for its function. Molecular genetic analysis using site-directed and deletion mutants revealed that loss of calmodulin binding completely abolished the function of DWF1 in planta, whereas partial loss of calmodulin binding resulted in a partial dwarf phenotype in complementation studies. These results provide direct proof that Ca2+/calmodulin-mediated signalling has a critical role in controlling the function of DWF1. Furthermore, we observed that DWF1 orthologues from other plants have a similar Ca2+/calmodulin-binding domain, implying that Ca2+/calmodulin regulation of DWF1 and its homologues is common in plants. These results raise the possibility of producing size-engineered crops by altering the Ca2+/calmodulin-binding property of their DWF1 orthologues.     

Clathrin light chains and secretory vesicle binding proteins are distinct

Recently, several groups have initiated studies on cytosolic proteins that bind to isolated secretory vesicle membranes in the presence of Ca2+ in order to identify proteins that may regulate exocytosis. Two major chromaffin granule binding proteins, of molecular weights 32,000 (32K) and 34,000 (34K), were reported to have the same mobility on one-dimensional SDS gels as clathrin-associated light chains from the adrenal medulla, and the 34K granule binding protein the same one-dimensional peptide map as the 34K clathrin light chain. These observations support the hypothesis that Ca2+-dependent recruitment of soluble light chains to the vesicle membrane may nucleate the assembly of a clathrin coat and initiate endocytosis. Here we report that two-dimensional peptide maps of the clathrin light chains and of all chromaffin granule membrane binding proteins in the 30K range are distinct, and therefore fail to support this hypothesis. It has also been suggested that some or all of the vesicle binding proteins require calmodulin for their interaction with the membrane. However, we find that antagonism of calmodulin by trifluoperazine does not prevent the association of the other cytosolic proteins with the chromaffin granule membrane.     

小麦耐盐突变体筛选及生理生化特性分析

以小麦幼穗、幼胚为原料材料,进行耐盐突变体的筛选,并对其愈伤组织及再生植株后代进行耐盐稳定性的生理生化分析,结果表明 （１）耐盐系在高盐浓度下其鲜重、干重明显高于对照系;（２）耐盐系保持较高的Ｋ＾＋／Ｎａ＾＋比;（３）对照系种子有１１条醇溶蛋白电泳带,而耐盐系为１４条,与对照系相比Ｂ２、Ｂ３、Ｂ４带为耐盐所特有,Ｂ１带含量高于对照物,但Ｂ５带含量低于对照系;（４）耐盐系再生植株后代可溶蛋白电泳带为     

小麦耐盐系筛选及稳定性研究

以小麦幼穗、幼胚为原始材料,进行耐盐突变体的筛选,并对其愈伤组织及再生植株后代进行耐盐稳定性的生理生化分析。结果表明：耐盐系在高盐浓度下其鲜重、干重明显高于对照系;耐盐系保持较高的Ｋ＾＋／Ｎａ＾＋比;与对照组相比,耐盐系种子醇溶蛋白电泳带有１４条,其中Ｂ２,Ｂ３,Ｂ４带为耐盐系所特有,Ｂ１带含量高于对照。但对照Ｂ５带合成量增加;耐盐系再生植株后代可溶蛋白电泳带耐盐系为２８条蛋白带,而对照系为２６条     

A calcium sensor in the sodium channel modulates cardiac excitability.

Sodium channels are principal molecular determinants responsible for myocardial conduction and maintenance of the cardiac rhythm. Calcium ions (Ca2+) have a fundamental role in the coupling of cardiac myocyte excitation and contraction, yet mechanisms whereby intracellular Ca2+ may directly modulate Na channel function have yet to be identified. Here we show that calmodulin (CaM), a ubiquitous Ca2+-sensing protein, binds to the carboxy-terminal 'IQ' domain of the human cardiac Na channel (hH1) in a Ca2+-dependent manner. This binding interaction significantly enhances slow inactivation-a channel-gating process linked to life-threatening idiopathic ventricular arrhythmias. Mutations targeted to the IQ domain disrupted CaM binding and eliminated Ca2+/CaM-dependent slow inactivation, whereas the gating effects of Ca2+/CaM were restored by intracellular application of a peptide modelled after the IQ domain. A naturally occurring mutation (A1924T) in the IQ domain altered hH1 function in a manner characteristic of the Brugada arrhythmia syndrome, but at the same time inhibited slow inactivation induced by Ca2+/CaM, yielding a clinically benign (arrhythmia free) phenotype.