自旋标记钙调蛋白与酸枣仁皂甙A相互作用研究

中草药有效成份酸枣仁皂甙A是钙调蛋白CaM的一种天然非竞争性拮抗剂。利用氮氧自由基马来酰亚胺衍生物标记CaM研究了二者的相互作用。结果表明,每分子CaM至少可以结合二个分子的酸枣仁皂钙A,二者的作用影响CaM上Lys残基主要是75,94)的环境,推测酸枣仁皂钙A是通过疏水作用结合到CaM分子两端的疏水沟区。通过比较三氟拉嗪TFP与酸枣仁皂甙A的结构特点,抑制性质与结合位点,提出了CaM调节环核苷酸磷酸二酯酶PDE的模式。     

钙调蛋白的自旋共振研究：拮抗剂和它的相互作用

利用碘乙酰胺氮氧自由基标记钙调蛋白研究了它与三氟拉嗪（ＴＦＰ）、酸枣仁皂甙Ａ（ＪｕＡ）的相互作用。结果表明,每分子ＣａＭ分别至少可以结合两分子的ＴＦＰ及ＪｕＡ,它们的作用影响了ＣａＭ上的Ｍｅｔ残基（主要是７１,７２和７６）的环境,使反应自由基运动自由度的旋转相关时间τＲ值下降。据τＲ变化的趋势,推测ＴＦＰ和ＪｕＡ都是通过疏水作用结合到ＣａＭ上的疏水沟区,但两者的结合位点可能不同。     

酸枣仁皂甙A与钙调蛋白相互作用的荧光光谱研究

文中报导了本实验室最近发现的一种新型钙调蛋白(CaM)天然拮抗剂——酸枣仁皂甙A,它能显著地抑制CaM活化PDE的活力.为研究它与CaM间的相互作用,本实验还制备了与天然CaM具有相同激活PDE能力的丹磺酰钙调蛋白(D-CaM).D-CaM的荧光光谱研究表明,酸枣仁甙A的加入诱导CaM分子的疏水位点更加暴露,从而增强丹磺酰基团的荧光发射量子产率.桔抗剂与CaM间的结合是绝对依赖Ca~(2 )的.荧光滴定的结果证明此结合的解离常数为2.8μM.酸枣仁皂甙A能进一步加强三氟啦嗪(TFP)所诱导的D-CaM荧光增强.这结果暗示,它不与TFP竞l争CaM上相同的结合位点.     

钙调蛋白抑制剂三氟拉嗪对海马脑片缺氧损伤的保护作用

为了研究钙调蛋白抑制剂三氟拉嗪(trifluoperazine, TFP)抗缺氧脑损伤的作用,应用海马脑片胞外记录技术和高效液相检测技术,研究了TFP对缺氧时脑片群峰电位(population spikes, PS)以及脑片孵育液中氨基酸含量的影响。结果表明, 50 滋mol/L TFP灌流的脑片,缺氧后PS的平均消失时间与缺氧组相比有显著差异(P<0.05);复氧后脑片PS的平均恢复程度为52.8%±14.3%,与缺氧组20.5%±9.8% 相比有非常显著差异(P<0.01);同时,TFP也可显著地抑制缺氧所致脑片孵育液中兴奋性氨基酸含量的增加(P<0.05)。由此可见,TFP对脑缺氧损伤有明显的保护作用,其作用机理可能与抑制海马组织兴奋性氨基酸的释放有关。     

钙调蛋白与拮抗剂TFP相互作用的水质子弛豫速率研究

钙调蛋白(CaM)是一种多功能调节蛋白,它对靶酶的作用受各种药物拮抗.三氟啦嗪(TFP)是CaM的一种有效拮抗剂.本文利用核磁弛豫法测定与TFP结合前后CaM中金属离子配位水的变化,从而推测CaM与TFP相互作用的方式.结合CD谱的测定结果,可以认为TFP与CaM结合摩尔比不超过2时是特异的强结合,但不影响金属结合部位及主链结构.当比例超过2时,额外的TFP以不同于前的方式结合于CaM.     

果蝇钙调蛋白和肌球蛋白NINAC相互作用分析

果蝇的视觉信号转导途径是已知的最快的G 蛋白偶联信号通路。这其中涉及到TRP/TRPL通道的开放以及钙离子的内流等一系列反应的形成。NINAC（neither inactivation nor afterpotential C）是一种特异性存在于果蝇感光细胞中的第3类肌球蛋白（Myosin III）,其在终止果蝇的视觉信号转导通路中起着非常重要的作用。NINAC蛋白具有两种亚型：一种是132 kD的蛋白亚型 (p132),另一种则是174 kD的蛋白亚型(p174)。这两种不同的蛋白亚型都具有相同的激酶催化结构域(kinase domain),以及与肌球蛋白相似的马达结构域(motor domain)。但是,它们在C末端却存在着非常大的差异,这其中包括了钙调蛋白结合基序(IQ motif)。NINAC的这两种蛋白亚型在果蝇的感光细胞中的定位以及作用有很大不同,尤其是在与钙调蛋白的相互作用方面。钙调蛋白结合基序与钙调蛋白（CaM）之间的相互作用对于果蝇的视觉信号通路具有重要的意义：NINAC结合钙调蛋白能力的缺失将导致果蝇的视觉传导缺陷。本文通过蛋白共表达的方法,成功表达并纯化得到了不同版本的NINAC与钙调蛋白的蛋白复合物。静态光散射的结果表明,在Ca2+存在情况下,p174蛋白可以结合2个Ca2+-CaM,而p132只结合1个Ca2+-CaM。通过分析型凝胶过滤以及等温量热滴定技术,进一步鉴定了p174及p132的IQ2（第2个钙调蛋白结合基序）序列与Ca2+ CaM的相互作用。通过序列分析及进一步的突变实验发现,p174 IQ2中的3个疏水氨基酸（F1083,F1086 和 L1092）对于钙调蛋白的结合非常重要,并导致了p174与p132蛋白和Ca2+ CaM结合能力的差异。本文的研究提供了NINAC与Ca2+-CaM相互作用的生化机制,将为进一步在果蝇视觉信号通路中深入研究CaM是如何调节NINAC的体内功能实验打下基础。     

铝与钙调蛋白相互作用的荧光光谱研究

本文研究了铝与钙调蛋白相互作用的荧光光谱。实验证明,Al~3与CaM的结合所引起的构象变化与Ca~(2+)与CaM结合所引起的构象变化既有相同之处,也有不同之处。Al~(3+)在CaM分子上的结合有特异性结合与非特异性结合两种情况。其特异性结合位点可能为2—3个。钙调蛋白的非竞争性拮抗剂酸枣仁皂甙A(JuA)可以继续抑制已被Al~(3+)部分抑制的PDE-CaM的活力。     

A Study on Spin-Labelled Oligonucleotide Synthesis and its Electron Spin Resonance Behavior in Solution

An oligonucleotide spin-labelled with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) at the internucleotide bond (d-Tp(L)TpTpTpT) prepared by oxidation of the pentanucleotide containing the H-phosphonate diester (d-Tp(H)TpTpTpT) in the presence of 4-amino-TEMPO, was separated and identified by high-performance, reverse-phase liquid chromatography combined with detection by electron spin resonance spectroscopy. This spin-labelled oligonucleotide produced a triplet with the slightly broadened M₁ = — I ESR component, while a triplet with almost equal intensities was obtained from the spin-label. The M₁ = —1 component from the labelled oligonucleotide was further broadened in the presence of poly(A) which forms a complementary double strand with this molecule.     

Comparative Analysis of Hair Melanins by Chemical and Electron Spin Resonance Methods

Eighteen hair samples from Karakul newborn lambs with various colors were estimated for eumelanin and pheomelanin contents (C_e and C_p, respectively) by electron spin resonance (ESR) spectrometry and by high-performance liquid chromatography (HPLC). Correlation coefficients between the values estimated by the ESR and HPLC methods were 0.96, 0.93, and 0.99 for C_e, C_p, and C_e/C_p, respectively. The high correlation coefficients show that both methods fit well for estimation of relative values of these parameters. The absolute values of C_e and C_e/C_p coincide rather well when C_e is high, but considerable discrepancies appear when C_e is low. The reasons for these discrepancies are discussed. The HPLC method appears to be more sensitive for detection of low concentrations of pheomelanin, while the ESR method fits well for mass selection purposes.     

Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum

The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2 M external Ni²⁺ to obtain spectra solely from labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50–60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3–8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5–2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.     

外源钙调蛋白对植物细胞分裂增殖作用的研究

外源钙调蛋白（Ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）对胡萝卜悬浮细胞增殖具明显促进作用,不同浓度ＣａＭ的促进程度不同,７ｕｇ／ｍｌ时促进作用最大。ＣａＭ抑制剂ＴＦＰ（Ｔｒｉｆｌｕｏｐｅｒ－ａｚｉｎｅ）则明显抑制该悬浮细胞的增殖,ＴＦＰ浓度越高则抑制作用越强。另外,外源ＣａＭ可以加快珍珠梅花粉第二次有丝分裂,改变生殖细胞有丝分裂各期花粉管的比例,说明外源ＣａＭ对植物体细胞和性细胞的增殖和分裂均有促进作用。     

钙调素高表达对NRK细胞中DG-PKC和cAMP-PKA水平的影响

钙调素（ＣａＭ）作为Ｃａ２＋的主要受体,对细胞增殖起重要调节作用,而且在转化细胞中ＣａＭ的水平明显高于正常细胞．ｃＡＭＰ作为一种第二信使,起着将细胞外刺激信号转化为细胞内各种生理活动的媒介作用．蛋白激酶Ａ（ＰＫＡ）则是这种转化过程中的关键激酶．蛋白激...     

Free Radicals in Rabbit Spinal Cord Ischemia: Electron Spin Resonance Spectroscopy and Correlation with SOD Activity

1. In nonanesthetized rabbits temporal occlusion of the abdominal aorta was used to induce oxidative stress in the lower part of the body including distal segments of the spinal cord.2. Spinal cord samples were taken from the animals exposed to 25-min aortic occlusion (AO ) or to occlusion followed by 1- or 2-hr reperfusion (AO/R1 or AO/R2, respectively) or from sham-operated animals (C). The presence of free radicals (FR) in the spinal cord samples frozen in liquid N₂ was assessed by ESR spectroscopy without spin trapping. Moreover, superoxide dismutase (SOD) activity and conjugated diene (CD) levels were measured in the samples.3. In the AO group FR were detected in the spinal cord regions close to the occlusion (lower thoracic and distal segments) along with a decrease in SOD activity. The calculated g value (g = 2.0291) indicated that the paramagnetic signal recorded might be attributed to superoxide radicals. FR were absent in the AO/R1 group. Concurrently, the SOD activity revealed a significant tendency to return to the control level. FR appeared again in the AO/R2 group, mostly in the upper and middle lumbar regions, along with a decrease in SOD activity. No sample from the C group revealed FR. A significant increase in CD levels was observed in the thoracolumbar region only in the AO/R2 group. The temporary absence of FR in the AO/R1 group suggests activation of defense antioxidant mechanisms (e.g., specific enzymatic systems such as SOD), which might have been exhausted later.4. Changes in SOD activity similar to those observed in the thoracolumbar region, though less noticeable, occurred in the obviously noncompromised tissue (upper cervical region). This points to a kind of generalized reponse of the animal to aortic occlusion.5. Direct ESR spectroscopy revealed the presence of FR as well as their time course in the spinal cord during the early phase of ischemia/reperfusion injury and the inverse relationship between FR and SOD activity.     

Electron paramagnetic resonance spectroscopy and molecular modelling of the interaction of myelin basic protein (MBP) with calmodulin (CaM)-diversity and conformational adaptability of MBP CaM-targets

The classic 18.5 kDa isoform of murine myelin basic protein (mMBP) has been shown to bind calmodulin (CaM) strongly and specifically in vitro. Here, we have used site-directed spin labelling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to map more precisely the sites of interaction of recombinant mMBP (rmMBP) with CaM. On the basis of these and previous experimental data, and the predictions of CaM-binding motifs using the Calmodulin Target Database (), three main segments of MBP were suggested for the interaction. The first site is located at the C-terminus; the second one lies in the central portion of the protein and forms an amphipathic alpha-helix in reconstituted myelin-mimetic systems; the third is quite close to the N-terminus. The murine Golli-MBP isoform J37 has also been shown to bind CaM in vitro, and an interaction site was predicted in the N-terminal Golli-specific portion of the protein. From these four segments, we selected peptide fragments of 12-14 residues in length, chosen on the bases of their amphipathicity and CaM-target characteristics. We modelled each of these peptides as alpha-helices, and performed docking simulations to investigate their interactions with the CaM peptide-binding tunnel. Different yet almost equally favourable CaM-binding modes were found for each of them. The experimental SDSL/EPR and theoretical modelling results were in good agreement, and supported the conjecture that there are several plausible CaM-binding sites in MBP, that could be induced into an alpha-helical conformation by their interaction with CaM and account for strong immobilisation of spin-labeled residues in all three segments. Phosphorylation and deimination were also emulated and simulated for known sites of MBP post-translational modification. The results obtained confirmed the appropriate utilisation of simple residue substitutions to mimic the natural modifications, and demonstrated molecular mechanisms by which MBP-CaM interactions could be modulated in vivo.