Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.     

乳酸对肌酸激酶活性抑制作用的研究

应用酶活测定的方法分析了肌酸激酶在不同浓度乳酸中的活性变化．实验结果说明在失活过程中没有蛋白质的聚沉；肌酸激酶的失活遵循一级反应的一相过程；失活与乳酸浓度和作用时间呈正相关．通过对比实验结果显示，乳酸是通过其解离状态的H^ 改变了机体PH值而影响肌酸激酶活性的，并且就乳酸对肌酸激酶影响及其意义进行探讨．     

Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

IntroductionArginine kinase ( EC 2 . 7. 3. 3,AK ) in inver-tebrates plays a central role in both temporal andspatial ATP buffering in cells with high,fluctuating energy requirements( muscle,nerves,etc.) by catalyzing the magnesium- dependentreversible phosphorylation between L- arginine andATP according to the following reaction[1,2 ]L- arginine+ Mg.ATP N- phospho- L- arginine+ Mg.ADP ( 1 )　 AK is an ideal paradigm for the classicalenzymology of bimolecular reactions[3 ,4] andactivit…     

NaCl对萝卜幼苗逆境指标及蛋白激酶活性的影响

研究了NaCl对萝卜(Raphanussativus)幼苗叶片内SOD,POD,CAT活性,Vc、脯氨酸、可溶性糖、蛋白质含量和蛋白激酶活性的影响以及激酶活性与部分金属离子的关系.结果表明,0 5%的NaCl能使萝卜体内SOD,POD,CAT活性,Vc、蛋白质含量以及生物量降低.与此同时,提高体内脯氨酸、可溶性糖,促进蛋白激酶活性增加,反应体系的Ca2 ,Mn2 ,Mg2 对蛋白激酶活性的发挥有显著影响.     

Rice receptor-like kinase OsSI-RLK2 inhibits internode elongation

Receptor-like kinase participates in the early events of plant signal transduction pathways. Previously, we screened the receptor-like kinase genes in rice and performed phylogenetic analyses. In this study, we isolated a receptor-like kinase gene, OsSI-RLK2, from rice. Expression of OsSI-RLK2 was induced by ABA treatment. In vitro analysis indicates that OsSI-RLK2 has Mn2 dependent autophosphorylation activity, but does not have this activity in the presence of Ca2 and Mg2 . Transgenic rice with over-expressed OsSI-RLK2 displayed shortened internodes resulting in a dwarf phenotype. Taken together, these results suggest that OsSI-RLK2 may represent a new type of functional RLK in rice that can inhibit the elongation of the internode.     

用点突变方法研究海参精氨酸激酶的活性位点

为研究动物能量代谢,用点突变的方法从双亚基海参(Stichopusjaponicus)精氨酸激酶中得到了3个突变体:C274A、R283G和H287A,并通过大肠杆菌E.coli表达。大部分精氨酸激酶都以包涵体的形式存在。经过纯化之后对3种突变体的催化活性和一些结构特征进行了分析。这3种突变体丧失了绝大部分催化活力,特别是突变体C274A,与野生型相比除了活性完全丧失之外,结构却几乎没有变化。这3个残基对海参精氨酸激酶结构或功能的保持有着重要作用,Cys274残基可能是海参精氨酸激酶的活性位点。     

大鼠实验性甲亢心肌内肌酸激酶变化的免疫组化研究

以药物诱导的方法建立了大鼠实验性甲亢模型，并以该模型心肌切片进行了免疫组化染色检测肌酸激酶。结果显示，随着甲亢病程的延长，着色呈现酶含量降低，酶活性减少的变化。实验证实甲亢时心肌呈现渐进性缺血过程。     

Activity and Structure Changes of Arginine Kinase from Shrimp Feneropenaeus chinensis Muscle in Trifluoroethanol Solutions

Trifluoroethanol has often been used in protein folding studies. The changes in activity and unfolding of arginine kinase from shrimp Feneropenaeus Ghinensis muscle during denaturation in different concentrations of trifuoroethanol were investigated using far-ultraviolet circular dichroism and fluorescence emission spectra. Arginine kinase was inactivated in trifluoroethanol solutions. The tertiary and secondary structures of arginine kinase were also destroyed in the trifluoroethanol solutions. The unfolding and inactivation courses were measured and compared. Inactivation occurred prior to unfolding, which suggests that the arginine kinase active site is more easily damaged by the denaturant than the enzyme as a whole. The result also indicates that the arginine kinase active site is situated in a limited and flexible region of the enzymemolecule.     

Ca2+ -CaM与乙烯调节草莓果实NAD激酶和NADP磷酸酶活性的关系

用外源乙烯单独以及乙烯分别与钙离子通道阻塞剂异博定(Verapamil,Vp)、钙调素拮抗剂氯丙嗪(Chloropromaize,CPZ)、三氟拉嗪(Trifluoperazine,TFP)处理乳白期草莓果实12 h,移去乙烯之后在空气中继续放置24h,测定果实乙烯释放率、NAD激酶活性及NADP磷酸酶活性的变化.结果表明,外源乙烯能诱导草莓果实乙烯大量合成,比对照(未经任何处理)提高420%和73%,抑制NAD激酶活性约20%和40%,对NADP磷酸酶影响不明显.Vp、CPZ和TFP均能逆转外源乙烯诱导的乙烯合成以及对NAD激酶活性的抑制作用,表明乙烯可能通过抑制草莓果实中NAD激酶活性从而促进和加快果实成熟衰老,Ca2 、CaM可能介导草莓果实的乙烯信号转导.     

天门冬氨酸对精氨酸激酶的作用研究

应用酶活测定、荧光发射光谱等方法分析了精氨酸激酶在不同浓度天门冬氨酸中活性变化和去折叠动力学。实验结果表明：天门冬氨酸引起精氨酸激酶失活与去折叠,并且过程中没有蛋白质的聚沉;精氨酸激酶的去折叠遵循一级反应动力学的二相过程,包括快相和慢相;失活与天门冬氨酸浓度呈正相关;当去除天门冬氨酸的影响,精氨酸激酶的活力能得到恢复。通过对比实验结果暗示,天门冬氨酸是通过其解离状态的H＋改变了机体pH值而影响精氨酸激酶活性的,并且就天门冬氨酸对精氨酸激酶影响及其意义进行探讨。     

丙酮酸、乳酸对体外肌酸激酶活性的抑制作用

 应用酶活测定的方法分析了肌酸激酶在不同浓度的乳酸、丙酮酸中的活性变化.实验结果说明在失活过程中没有蛋白质的聚沉,失活与乳酸浓度、丙酮酸的浓度呈正相关.通过对比实验结果提示,丙酮酸离子、乳酸离子对肌酸激酶没有明显影响,丙酮酸、乳酸是通过其解离状态的H⁺改变了机体pH值而影响肌酸激酶活性的,作者就丙酮酸、乳酸对肌酸激酶影响及其意义进行探讨.     

Effects of Glycerol in the Refolding and Unfolding of Creatine Kinase

IntroductionProtein folding is the process by which the aminoacid sequence of a protein determines the three-dimensional conformation of the functionalprotein[1 ] . The elucidation of the molecularmechanism of protein folding from a disorderedpolypeptide chain to the specific native stateremains one of the major challenges inbiochemistry[2 ,3] ,namely,deciphering the secondhalf of the genetic code[4 ] .Molecular chaperonesplay an importantrole in protein folding in vivo aswell asin vitro.A cha…     

HeLa细胞中激酶NUAK1调控Tuberiun的磷酸化

NUAK1是LKB1的下游激酶之一,可被LKB1磷酸化而激活,但对其在LKB1相关信号通路中的功能仍缺乏了解.本研究发现在HeLa细胞中重建LKB1表达后NUAK1与tuberin蛋白免疫共沉淀,提示在野生型LKB1存在时NUAK1与tuberin可能存在直接相互作用.进一步的激酶活性测定和in vivo蛋白磷酸化实验表明:在HeLa细胞中葡萄糖匾乏条件下,野生型LKB1可显著激活NUAK1的激酶活性;而被激活的NUAK1可明显提高tuberin 的磷酸化水平,使用NUAK1 siRNA pool干扰NUAK1的表达则几乎将tuberin的磷酸化水平降低为零.上述结果表明NUAK1可能介导了LKB1对tuberin磷酸化的调节,进而下调mTOR通路,抑制蛋白质合成与细胞生长增殖.     

重组酵母Saccharom yces cerevisiae蛋白激酶Sch9的分泌表达与活性鉴定

为了深入研究蛋白激酶Sch9的生物化学特性,在Pichia pastoris酵母KM71H菌种中分泌表达并纯化了重组Sch9蛋白.进一步通过体外磷酸化实验分析了重组Sch9蛋白的蛋白激酶活性.通过重组蛋白质工程的手段初步研究了Sch9蛋白的生物化学和酶学特性.     

尘螨肠道微生物蛋白核苷二磷酸激酶的克隆、表达及纯化

根据 GenBank 中 NDP kinase 的基因序列,采用生物信息学方法将其中稀有密码子改造为大肠埃希菌常用密码子并进行二级结构优化,合成 NDP kinase 基因,构建原核表达载体 pET28a-NDP kinase 并酶切鉴定其序列,在大肠埃希菌 BL21（DE3）中用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,重组产物采用镍离子金属螯合亲和层析柱纯化.经密码子改造和二级结构优化后 NDP kinase 基因长度为490 bp,其编码蛋白理论分子量为21.5 kDa,重组表达载体经酶切鉴定与理论推测结果相符,在大肠埃希菌 BL21（DE3）中该基因经 IPTG 诱导可高效表达,纯化后的重组蛋白分子量约为21.5 kDa,其单一蛋白纯度达95;以上.本研究成功构建了尘螨肠道微生物蛋白核苷二磷酸酶（NDP kinase）基因的 pET28a 原核重组质粒,为进一步研究 NDP kinase 在尘螨疫苗免疫治疗中的作用机理提供了基础.     

Roles of MAP kinase signaling pathway in oocyte meiosis

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases expressed widely in eukaryotic cells. MAPK is activated by a cascade of protein kinase phosphorylation and plays pivotal roles in regulating meiosis process in oocytes. As an important physical substrate of MAPK, p90^rsk mediates numerous MAPK functions. MAPK was activated at G2/M transition during meiosis. Its activity reached the peak at MⅠ stage and maintained at this level until the time before the pronuclear formation after fertilization. There is complex interplay between MAPK and MPF in the meiosis regulation. Furthermore, other intracellular signal transducers, such as cAMP, protein kinase C and protein phosphotase, ect., also regulated the activity of MAPK at different stages during meiosis in oocytes. In the present article, the roles of MAPK signaling pathway in oocyte meiosis are reviewed and discussed.     

混合拥挤试剂对人肌肌酸激酶稳定性的影响作用研究

为了更加真实地模拟细胞内环境,在人肌肌酸激酶(HCK)的去折叠环境中同时加入了大分子拥挤试剂(葡聚糖)和小分子渗透剂(蔗糖),采用酶活性、荧光发射谱和溶液浊度检测等方法研究肌酸激酶在含有蔗糖和葡聚糖的混合拥挤环境中的稳定性,结果表明在肌酸激酶盐酸胍变性过程中,蔗糖不仅抑制肌酸激酶酶活性的丧失,还稳定肌酸激酶的构象,而葡聚糖对肌酸激酶保护能力相对较弱.对于热变性过程,相同质量浓度的蔗糖和葡聚糖能同等程度地减缓肌酸激酶活性的丧失,并抑制聚沉的产生.在葡聚糖和蔗糖同时存在的情况下,两者的保护作用可以累加,并共同维持肌酸激酶的稳定性.     

大豆叶片57kD钙依赖蛋白激酶自磷酸化性质的研究

本实验证明大豆叶片质膜上57kD钙依赖蛋白激酶具有较强的体外自磷酸化活性；并且其体外自磷酸化水平受到人工诱导衰老处理的明显促进及外源6－BA预处理的有效抑制，暗示该激酶可能参与外源细胞分裂素对大豆叶片衰老的调控过程。进一步的生化分析表明，其自磷酸化位点可能发生在丝氨酸和苏氨酸等残基上，而在酷氨残基上未发生磷酸化反应。     

葡萄糖酸钙对肌苷发酵的影响

在肌苷的摇瓶发酵过程中,10g／L的葡萄糖酸钙适于肌苷的合成和菌体生长。初始培养基中加入10g／L的葡萄糖酸钙,能够诱导葡萄糖酸激酶的生成,大幅提高其比活,增大磷酸戊糖（HMP）途径的通量。肌苷的产率由10.76g／L提高到18.36g／L。     

抗三尖杉酯碱的HL60细胞蛋白质磷酸化

研究了抗三尖杉酯碱的ＨＬ６０细胞蛋白质磷酸化的变化。经差速离心得到纯膜蛋白，抗性细胞有一高度磷酸化的１１０ｋｕ的蛋白质存在，免疫沉淀ｃ－ＫＡＦ－１蛋白激酶，抗性细胞内ｃ－ＲＡＦ－１蛋白激酶磷酸化程度明显提高，其活性被蛋白激酶Ｃ抑制剂ＣａｌｐｈｏｓｔｉｎＣ明显地抑制。结果表明：ＨＬ６０细胞对三尖杉酯碱的抗药性与蛋白质高度磷酸化有关；抗性细胞内ｃ－ＲＡＦ－１蛋白激酶磷酸化程度明显提高，可能与多药抗药性和抗细胞调亡有关。