酷蛋白一级结构特征及酷蛋白胶粒新模型

αｓ１、β、αｓ２酪蛋白中磷酸化Ｓｅｒ周围序列具有Ｓｅｒ－Ｓｅｒ－Ｓｅｒ－Ｇｌｕ－Ｇｌｕ的共同特点和ｃＡＭＰ、ｃＧＭＰ、Ｃａ＾＋＋调节的蛋白质的磷酸化Ｓｅｒ周围序列和Ｈｓｐ７０钙调蛋白结合序列具有相似性，都具有２－３个Ｓｅｒ相连的序列，不同之处在于Ｎ端的碱性氨基酸簇变成了Ｃ端的Ｇｌｕ－Ｇｌｕ，反映了用于携带Ｃａ＾＋＋和调节作用的Ｓｅｒ磷酸化二类蛋白质一级结构的差别：前者需要有一个酸性残基区协助，后     

Tb（Ⅲ）与DTPAA,pAS—Na的配合物在水溶液中荧光性质？…

用ＴｂＣｌ３,二乙三胺五乙酸二酸酐（简写为ＤＴＰＡＡ）和对－氨基水杨酸的钠盐（简写为ｐＡＳ－Ｎａ）在水溶液中制得Ｔｂ＾３＋的三元配合物,通过紫外可我谱和荧光光谱验证了此三元配合物的存在;用ＤＴＰＡＡ与ｐＡＳ－Ｎａ制得的含氮多羧基配体ＤＴＰＡ－ｐＡＳ和ＤＴＰＡ－２ｐＡＳ分别与ＴｂＣｌ３溶液制得了２种二元配合物。研究了此３种配合物在水溶液中的荧光性质,考察了浓度、ｐＨ值、Ｌａ＾３＋离子等对三元配合物荧     

高氯酸铽、六氟磷酸铽与二苯基亚砜、苯丁基亚砜配合物的发光

合成了Ｔｂ（ＤＰＳＯ）６·（ＣｌＯ４）３、Ｔｂ（ＤＰＳＯ）７·（ＰＦ６）３及Ｔｂ（ＰＢＳＯ）６·（ＣｌＯ４）３配合物（ＤＰＳＯ为二苯基亚砜ＰＢＳＯ为苯丁基亚砜）．在室温下测定了它们的固体粉末荧光激发和发射光谱，结果表明Ｔｂ（ＤＰＳＯ）６·（ＣｌＯ４）３配合物中Ｔｂ３＋的发光强度最高，其中５Ｄ４→７Ｆ５跃迁谱带强度高达８０６．２ｃｄ，Ｔｂ（ＤＰＳＯ）７（ＰＦ６）３配合物中Ｔｂ３＋发光强度次之，Ｔｂ（ＰＢＳＯ）６（ＣｌＯ４）３配合物中Ｔｂ３＋的发光强度最弱．这说明二苯亚砜做为配体受激后向Ｔｂ３＋离子传递能量的效率高于苯丁基亚砜，另外ＣｌＯ４－做为阴离子也可增加Ｔｂ３＋的发光强度，原因是部分外界ＣｌＯ４－离子可进入内界参予配位，降低了配合物的对称性，部分解除了跃迁禁律，提高了Ｔｂ３＋的发光强度．     

兔角膜上皮特异性水溶性蛋白质的电泳分析

分离兔角膜上皮和皮肤表皮，提取水溶性蛋白质，采用ＳＤＳ－ＰＡＧＥ以及ＩＥＦ／ＳＤＳ－ＰＡＧＥ技术对其组成进行分析研究，发现兔角膜上皮存在５种主要特异性水溶性蛋白质，其分子量和等电点分别为：Ｍｒ６８２００，ｐＩ７．５；Ｍｒ６１７００，ｐＩ５．１；Ｍｒ５１３００，ｐＩ７．６；Ｍｒ３６３００，ｐＩ８．９和Ｍｒ２５７００，ｐＩ５．７。     

狗脑抗吗啡镇痛肽“60a”的分离纯化及部分一级结构

本实验室从正常狗脑中又分离获得一个暂称这为“６０ａ”的肽。冻干粉经ＳｅｐｈａｄｅｒＧ－５０和羧甲基纤维素ＣＭ２２柱层析，再经反相高效液相层析纯化得最后样品，经ＳＤＳ电泳和等电聚焦电泳鉴定均为一条带，分子量约８０００，等电点在ｐＨ７．８左右；Ｎ－末端氨基酸为Ｍｅｔ；自Ｎ－末端的部分氨基酸序列为：Ｈ２Ｎ－Ｎｅｔ－Ｌｅｕ－Ｓｅｒ－Ｐｒｏ－Ａｌｓ－Ａｓｐ－Ｌｅｕ－Ｔｈｒ－Ａｓｐ－Ｉｌｅ－Ｌｅｕ－Ｐｈｅ－Ｔ     

背角无齿蚌碱性磷酸酶的功能基团研究

在一定条件下，分别采用ＰＭＳＦ，ＤＴＴ，ＰＣＭＢ，ＮＢＳ，ＴＮＢＳ，ＳＵＡＮ，ＢｒＡｃ及ＩＡｃ等化学修饰剂，选择修饰背角无齿蚌碱性磷酸酶的多种氨基酸残基，并测定其酶活力变化。结果表明，ＰＭＳＦ，ＮＢＳ，ＴＮＢＳ，ＳＵＡＮ和ＤＴＴ的修饰能显著抑制酶的活力，而且活力的降低程度与修饰剂的浓度相关。ＢｒＡｃ，ＩＡｃ和ＰＣＭＢ的修饰不表现对酶的抑制作用。初步认为，Ｓｅｒ，Ｌｙｓ和Ｔｒｐ残基是背角无齿蚌碱性磷     

稀土离子（Ⅲ）与牛血清白蛋白的相互作用Ⅰ．结合反应热力学

在ｐＨ６．３０、３７℃条件下，采用离心超过滤技术测定了１１种稀土离子（Ⅲ）与牛血清白蛋白（Ｂ８Ａ）的专一性结合常数Ｋ＿ｓ和非专一性结合常数Ｋ＿（ｎｓ）观察到Ｋ＿ｓ随稀土系列原子序数（Ｚ＿（ＲＥ））的变化有明显的＂四分组效应＂和＂钆断现象＂，而Ｋ＿（ｎｓ）～Ｚ＿（ＲＥ）类似于一元羧酸－稀土配合物稳定常数的变化规律。求出Ｔｂ（Ⅲ）－ＢＳＡ结合反应的热力学函数分别为ΔＨ＿ｓ＝４４．５±０．８ｋＪ／ｍｏｌ，ΔＳ＿ｓ＝２４３．２±２．４Ｊｍｏｌ·Ｋ，ΔＨ＿（ｎｓ）＝１９．９±１．１ＫＪ／ｍｏｌ，ΔＳ＿（ｎｓ）＝１４１．４±３．２Ｊ／ｍｏｌ·Ｋ。根据不同ｐＨ下测得的Ｔｂ（Ⅲ）与ＢｓＡ结合的Ｋ＿ｓ和Ｋ＿（ｎｓ）拟合出ＢＳＡ分子中参与稀土配位的基团的质子解离常数ｐＫ＝４．６０，进而确定出这些基团主要为天冬氨酸和谷氨酸残基侧链中的β、γ－ＣＯＯ￣－。     

Pyrococcus sp.DNA聚合酶内蛋白子突变体的剪切性质

将含麦芽糖结合蛋白－嗜热菌ＤＮＡ聚合酶内蛋白子基因的ｐＭＩ８４、ｐＭＩ９４、ｐＭＩ９５三个重组突变体质粒转入Ｅ．ｃｏｌｉ２４２６经发酵及ＩＰＴＧ低温诱导表达,直链淀粉糖亲和纯化获得ＭＩ８４（ｓｅｒ１／Ｓｅｒ５３８Ｓｔｏｐ）、ＭＩ９４（Ｓｅｒ１→ＣｙｓＨ１／Ｓｅｒ５３８Ｓｔｏｐ）、ＭＩ９５（Ｓｅｒ１→Ａｌａ１／Ｓｅｒ５３８Ｓｔｏｐ）三种蛋白质前体。研究了ＰＨ温度、巯基试剂对内蛋白子Ｎ末端的剪切影响。     

稀土硝酸盐与5-Br-PADAP固体配合物的合成及性质研究

在丙酮中合成出部分镧系硝酸盐与５　Ｂｒ－ＰＡＤＡ Ｐ 的固态配合物Ｌｎ（５—Ｂｒ— ＰＡＤＡＰ），（ＮＯ３）３·ｎＨ２Ｏ（Ｌｎ＝Ｌａ．Ｈｏ，Ｔｍ，ｎ＝１；Ｌｎ＝Ｓｍ，Ｅｕ，Ｇｄ，Ｔｂ，ｎ＝ ２）．屋过元素分析．红外光谱，紫外－可见光谱，热分析，Ｘ光粉末衍射物相分 析．溶解性及摩尔电导等测定．研究了配合物的有关性质。     

AS—3毒素的分离纯化及理化性质研究

ＡＳ－３毒素是由作者分离鉴定的一株梭关芽孢杆菌产生的神经性毒素，是一种大分子蛋白质。作者对ＡＳ－３毒素的分离纯化及理化性质作了初步研究，测得其蛋白质分子由两个亚基组成，分子量为１４８０００Ｄａ，等电点为５．９６，最大吸收波长２８８．５ｎｍ，含１７种氨基酸，二级结构中α－螺旋占多数，β－折迭和无规则卷曲较少。     

Recombinant PsbF from Synechococcus sp. PCC 7002 forms β:β homodimeric cytochrome b559

All organisms with oxygenic photosynthesis contain two photosystems:photosystem Ⅰ(PSⅠ)and photo-systemⅡ(PSⅡ),The minimal photosystem Ⅱ particles which are photochemically active contain three subunits:D1,D2 and cytochrome b559 (Cyt b559),The function of Cyt b559 remains unclear,We have successfully overxpressed the psbF gene,encoding the β subunit of Cyt b559,from a marine cyanobacterium Synechoccous sp.PCC 7002 as a fusion gene and obtained a redox-active from of Cyt b559,When the N-terminal GST protein of the fusion gene product was removed with thrombin ,the PsbF protein was still redox-active,suggesting that the recombinant PsbF can form dimer in Escherichia coli.The absorption spectra of either the oxidized from or the reduced form of both GST fusion protein and the purified PsbF dimer and the difference Spectra between the two forms are the same as that of the Cyt b559 isolated from the higher plants .Redox titration analysis of recombinant PsbF showed that the mid-point redox potential of the recombinant Cyt b559 was approximately 50 mV, which is close to the low potential of Cyt b559 ,The results are helpful to the understanding of locatlization and function of Cyt b559 on thylakoid membranes.Ⅰ     

Effect of impulse blockage on cytochrome oxidase activity in monkey visual system

Cytochrome oxidase (cytochrome c oxidase; ferrocytochrome c: oxygen oxidoreductase, EC 1.9.2.1) has been introduced as an oxidative metabolic marker for neurones in the central nervous system. Previous studies have shown that mature neurones remained sensitive to altered functional demands, and that both developing and adult neurones responded to sensory deprivation or deafferentation by reducing their cytochrome oxidase (Cyt. Ox.) activity. More recently, we showed that the blockage of retinal impulse transmission with tetrodotoxin led to a reversible reduction in Cyt. Ox. staining of affected lateral geniculate (LGN) and striate neurones in adult cats. The present study sought to extend these findings to adult monkeys, where Cyt. Ox. 'puffs' or 'blobs' are uniquely present in the visual cortex. We found that, while the retina remained histologically intact, with only moderate decreases in Cyt. Ox. staining of large ganglion cells and the two plexiform layers, subtle changes occurred in the LGN as early as 1 day post-tetrodotoxin injection, and clear reduction in enzyme levels was evident in both the LGN and the visual cortex by 3 days. Changes became progressively more severe up to 4 weeks post-injection. Within area 17, alternating bands of high and low Cyt. Ox. staining occurred in lamina IV, with alternating rows of dark and lightly reactive puffs superimposed in exact register. Thus, the mature visual neurones in the primate remain extremely sensitive to the cessation of retinal impulse transmission, and plastic metabolic changes occur through several synapses along the sensory pathway.     

Biosensors based on acetylcholinesterase immobilized on mesoporous silica thin films

Using tetraethoxysilane and 3-aminopropyltriethoxysilane as the silica sources, amino-functionalized mesoporous silica thin films with 3-dimensional cubic structure have been deposited on conducting ITO substrate in the presence of surfactant F127 templates under acid conditions. The acetylcholinesterase （AchE） and cytochrome c （Cyt c） were incorporated into the pores of mesoporous thin films, and an amperometric biosensor was obtained. After adsorption of AchE and Cyt c, the ordered cubic structure of mesoporous silica and the bioactivities and electrochemical activities of the immobilized protein and enzyme molecules were retained. The sensor properties of the biosensor were investigated by using acetylthiocholine iodide as the substrate and Cyt c as the electron transfer mediator. The inhibition versus the logarithm of concentration was found to be linear to organophosphorus pesticide dichlorvos over the concentration ranges of 1.0×10-8 mol/L to 1.0×10-3 mol/L with the detection limit of 3.1×10^-9 mol/L.     

Au@SiO2修饰电极的制备及对Cyt c的直接电化学研究

研究Au@SiO₂复合纳米材料修饰电极的制备方法及其对细胞色素c (Cyt c)的电催化行为.Cyt c在该修饰电极上表现出一对氧化还原峰，且在1.0×10^-7~2.0×10^-5 mol/L浓度范围内，Cyt c峰电流与浓度呈良好的线性关系，检测下限为5.0×10^-8 mol/L (S/N=3).还应用紫外可见光谱考察了Au@SiO₂复合纳米材料的生物兼容性，结果表明，Au@SiO₂材料为Cyt c提供了一个“近水”的微环境，使其在该修饰电极表面不易变性，因而具有良好的生物相容性.     

Electrostatic effect on electron transfer between cytochrome b5 and cytochrome c

The binding and electron transfer between wild type, E44A, E56A, E44/56A, E44/48/56A/D60Aand F35Y variants of cytochrome b5 and cytochrome c were studied. When mixed with cytochrome c, the cytochrome b5E44/48/56A/D60A did not show the typical UV-vis difference spectrum of absorption, indicating that the alteration ofthe surface electrostatic potential obviously influenced the spectrum. The electron transfer rates of wild type cytochromeb5, its variants and cytochrome e at different temperature and ionic strength exhibited an order of F35Y > wild type >E56A > E44A > E44/48/56A/D60A. The enthalpy and entropy of the reaction did not change obviously, suggestingthat the mutation did not significantly disturb the electron transfer conformation. The investigation of electron transfer rateconstants at different ionic strength demonstrated that electrostatic interaction obviously affected the electron transfer pro-cess. The significant difference of Cyt b5 F35Y and E44/48/56A/D60A from the wild type protein further confirmed thegreat importance of the electrostatic interaction in the protein electron transfer.     

社鼠和针毛鼠线粒体DNA序列的歧异及其系统进化关系

社鼠和针毛鼠是有着广泛地理分布的山地鼠种,它们的一些形态特征及核型明显有别于同属的其它鼠类.社鼠的亚种--雷琼社鼠局限于分布在广东省西部地区和海南省,它的形态特征多似社鼠,但也有少数特征相似于针毛鼠.对于社鼠和针毛鼠的分类地位及社鼠亚种的分化一直有着各种讨论.本研究进行了社鼠和雷琼社鼠以及针毛鼠龙门种群和香港种群的细胞色素b (264个核苷酸位点)和12S rRNA (387个核苷酸位点)基因片段的测序.社鼠与雷琼社鼠线粒体的细胞色素b基因片段间包含有19个变异位点、12S rRNA 基因片段间包含11个变异位点和2个插入/缺失位点.针毛鼠的2个种群间的细胞色素b 和12S rRNA 基因片段分别包含了3个和11个变异位点.通过邻接法和最大简约法重建的系统进化关系表明:雷琼社鼠与社鼠有着紧密的亲缘关系,它的亚种地位得到了线粒体DNA序列证据的肯定.针毛鼠龙门种群和香港种群细胞色素b和12S rRNA 基因片段序列的歧异反映出两个种群由于长期的地理隔离及其所经历自然选择的结果.     

Electrostatic effect on electron transfer between cytochrome b5 and cytochrome c

The binding and electron transfer between wild type, E44A, E56A, E44/56A, E44/48/56A/D60A and F35Y variants of cytochrome b5 and cytochrome c were studied. When mixed with cytochrome c, the cytochrome b, E44/48/56A/D60A did not show the typical UV-vis difference spectrum of absorption, indicating that the alteration of the surface electrostatic potential obviously influenced the spectrum. The electron transfer rates of wild type cytochrome bj, its variants and cytochrome c at different temperature and ionic strength exhibited an order of F35Y > wild type > E56A > E44A > E44/48/56A/D60A. The enthalpy and entropy of the reaction did not change obviously, suggesting that the mutation did not significantly disturb the electron transfer conformation. The investigation of electron transfer rate constants at different ionic strength demonstrated that electrostatic interaction obviously affected the electron transfer process. The significant difference of Cyt b, F35Y and E44/48/56A/D60A from the wild type protein further confirmed the great importance of the electrostatic interaction in the protein electron transfer.     

HRP标记的二抗检测heme结合蛋白造成假阳性问题的探讨

为了验证对辣根过氧化物酶(horseradish peroxidase, HRP)作为二抗标记物在检测heme结合蛋白时产生假阳性问题的推测,以菠菜细胞色素b_6f复合体(Cyt b_6f)和铁氧还蛋白-NADP~+氧化还原酶(FNR)为检测对象(前者含有heme结合蛋白Cyt f,后者不含heme基团),采用菠菜FNR一抗、HRP和碱性磷酸酶(alkaline phosphatase,ALP)标记的二抗,设计对比实验。结果表明,在使用HRP标记的二抗体系中,Cyt f不经一抗和二抗孵育即可显色,这是明显的假阳性;而在ALP标记的二抗体系中,Cyt f条带不显色,只有FNR条带显色。在western blot实验中,如果被检测蛋白为heme结合蛋白或者混有少量heme结合蛋白,应避免采用HRP标记的二抗,建议使用ALP标记的二抗或者其他二抗产品;heme结合蛋白具有类似HRP的氧化还原酶活性,其本身可以和显色液反应,从而产生假阳性,干扰对实验结果的判断。     

发酵乳杆菌β-半乳糖苷酶转糖基活性研究

从传统发酵乳制品中筛选到1株转糖基活性较高的乳杆菌,经16S rDNA菌种鉴定为发酵乳杆菌（Lactobacillus fermentum, EU621851）K4。以乳糖为底物,研究β-半乳糖苷酶粗酶液在不同pH、乳糖浓度、反应温度和时间的转糖基活性。结果表明在pH 5.5、乳糖20%、温度50℃条件下反应48h,转糖基活性最高,能生成多种低聚半乳糖。研究发现在低乳糖含量（5%）和pH 5～7范围内都具有明显的转糖基活性。用牛奶为原料进行转糖基能力测定,结果该酶既能降低牛乳中的乳糖含量,又能生成低聚半乳糖。因此,菌株Lb. fermentum K4在乳制品发酵中具有潜在的应用价值。     

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b 5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b 5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b 5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b 5 with cytochrome c are: wild type K A=4.2(±0.01)×10 6(mol/L) -1 , Phe35Tyr K A=3.7(±0.01)×10 6(mol/L) -1 and Phe35Leu K A=4.7(±0.01)×10 6(mol/L) -1 respectively ( I =1 m mol/L, pH 7.0 soldium phosphate buffer, 25℃). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b 5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b 5 and cytochrome c.