Nonlabens dokdonensis视紫红质2中的钠离子结合与pH依赖性的传递（英文）

正Dear editor,Light-driven Na~+ pumping rhodopsins(Na Rs),the next generation optogenetic tools,are a family of seven-helical transmembrane proteins and covalently link to a retinal chromophore~([1]).Na Rs have a putative Na~+ -conducting motif of Asn112-Asp116-Gln123~([1]),homologous to the H~+-conducting motif of Asp85-Thr89-Asp96 in H~+ pump bacteriorhodopsin(BR)~([2]).     

昆虫钠离子通道的研究进展

昆虫只有一个或两个电压门控钠离子通道α亚基基因,但两种转录后修饰(选择性剪切和RNA编辑)实现了昆虫钠离子通道的功能多样性.昆虫β辅助亚基TipE和TEH1-4在钠离子通道表达和调控中也起着重要作用.电压门控钠离子通道在动作电位的产生和传递中至关重要,是多种天然和人工合成神经毒素及杀虫剂的作用靶标,包括广泛使用的拟除虫...     

MiyabenolC和KobophenolA与雌激素受体的结合位点

iyabenolC (MiyC)和kobophenolA (KobA)是两种新型的植物雌激素。为了探讨MiyC和KobA与雌激素受体 (ER)的结合部位 ,运用计算机辅助分子模拟构建它们与ER结合的空间模型 ,找出结合位点 ,设计ER的两个突变体M1ER(ERM517AG52 1D)和M2ER(ERE353GR394 G) ;运用PCR技术将ER与MiyC或KobA的结合位点进行突变 ;运用报告基因检测实验 ,检测MiyC和KobA对突变的ER是否具有激活功能。结果显示MiyC激活M1ER使之促下游基因转录的作用下降 ,KobA对M1ER无激活作用 ;MiyC和KobA对M2ER无激活作用。以上结果显示MiyC和KobA与ER的结合位点可能为ER的Glu353 、Arg394 、Met517和Gly52 1。     

水稻NACA基因家族的鉴定及NACA2功能研究

该研究从水稻中鉴定了5个编码NACA蛋白的基因,并对其理化性质、结构、定位及表达进行分析,并针对NACA2的亚细胞定位及其在抗旱过程中的生物学功能进行了初步研究。结果表明：(1) 5个水稻NACA蛋白的氨基酸序列中含有多个保守的基序,NACA1-NACA4均含有NAC结构域和UBA结构域,但NACA5序列较短且不含UBA结构域;不同植物NACA蛋白的氨基酸序列进化关系与物种之间的进化关系高度统一。(2)组织表达模式分析发现,NACA1、NACA2和NACA3在不同水稻组织中具有较高的表达水平,尤其是在生殖器官中,但NACA4和NACA5在不同组织中的表达水平均很低;NACA基因的表达受到甘露醇、脱落酸(ABA)、茉莉酸(JA)、氯化钠(NaCl)、水杨酸(SA)及低温胁迫的诱导,其中NACA2和NACA5的表达变化最明显。(3)亚细胞定位表明,NACA2蛋白定位于细胞核和细胞质中。(4)与野生型相比,过表达NACA2拟南芥株系的萎蔫程度较轻,颜色较绿,显著降低了叶片的失水速率,且复水速度明显更快,并显著增强了对干旱和渗透胁迫的抗性。研究表明,NACA2正调控植物对干旱胁迫的抗性,为进一...     

MS2噬菌体衣壳蛋白与包装位点结合特异性及其生物学应用进展

MS2噬菌体为正义单链RNA噬菌体,基因组含有3569个核苷酸,编码成熟酶蛋白、衣壳蛋白、复制酶蛋白和裂解蛋白。MS2噬菌体复制酶编码基因5'端一个由19个碱基组成的茎环结构(又称包装位点)是衣壳蛋白二聚体与RNA相互作用的部位,二者相互作用形成的复合物是启动噬菌体自我包装的信号。MS2噬菌体衣壳蛋白与包装位点结合的特异性已被应用于RNA病毒核酸检测的标准物质、校准品和质控品的研究,实时动态监测活细胞内RNA的运动,以及RNA体内递送载体的研究等领域。     

悬浮培养桐叶槭细胞中NPA结合位点的特性与功能

在桐叶槭(Acer pseudoplatanus L.)悬浮培养细胞的膜组分上有 NPA(N-1-naphthylpathalamic acid)专一结合位点。它与NPA有高亲和力。它们的平衡解离常数(Kd值)为 7.5×10~(-9)mol/L。在4℃保温 60分钟,NPA的结合量达最高点,平衡状态至少可维持2小时。人工合成的生长素类物质:1-NAA(1-萘乙酸)、2-NAA、2,4-D以及生长素运输抑制剂2,3,5-TIBA(三碘苯甲酸)均能与NPA竞争同一结合位点。这些物质与此结合位点的Kd值范围为 10~(-4)—10~(-6)mol/L之间。实验表明:NPA结合位点可能在调节细胞内IAA水平上起作用,从而影响细胞的生长。     

Dppa2 基因启动子区Oct4 结合位点突变对其活性的影响

目的：探讨Dppa2 基因5'' 端启动子区Oct4 结合位点突变对Dppa2基因启动子活性的影响。方法：PCR 扩增包括Oct4 结合 位点的Dppa2 基因5'' 端转录起始点上游-2439～+293 bp 的启动子序列,片段长度为2732 bp。将该片段连接到pGL3-Basic 载体, 构建野生型pGL3-2439表达载体。采用定点突变法,将-1959～-1957 位碱基的GCA突变成TAG,构建Oct4 结合位点突变型 pGL3-mo2439 表达载体。用上述两种表达载体、PGL3-basic 载体和Oct4 表达载体分别瞬时转染HEK 293 细胞。细胞培养48 h 后,利用双荧光素酶报告系统测定各组细胞表达的荧光素酶的相对活性。结果：经琼脂糖凝胶电泳及测序鉴定,证实野生型 (pGL3-2439)和突变型(pGL3-mo2439)载体构建成功。荧光素酶活性测定结果显示,转染Dapp2 基因启动子野生型pGL3-2439 表 达载体的细胞组荧光素酶的相对活性为16.307,突变型pGL3-mo2439 表达载体的细胞组荧光素酶的相对活性为10.634。Oct4 结 合位点突变后,Dppa2 基因启动子区转录活性较野生型降低了35 %。结论：Dppa2基因5''端启动子区-1959～-1957 位的Oct4 结 合位点突变可能导致Dppa2 基因启动子活性下降     

TANK结合激酶1在PINK1/Parkin依赖性和非依赖性线粒体自噬中的作用研究进展

线粒体自噬是一种清除受损或多余线粒体的过程,在调节细胞内线粒体质量和维持线粒体能量代谢等方面发挥重要作用。TANK结合激酶1 (TANK-binding kinase 1, TBK1)是一种多功能的丝氨酸/苏氨酸蛋白激酶,同时参与调控PTEN诱导假定激酶1 (PTEN-induced putative kinase 1, PINK1)/Parkin依赖性和非依赖性线粒体自噬过程。近期研究表明,TBK1可磷酸化视神经蛋白(optineurin, OPTN)、p62/sequestosome-1、Ras相关GTP结合蛋白7 (Ras-related GTP binding protein 7, Rab7)等自噬相关蛋白,并介导核点蛋白52 (nuclear dot protein 52, NDP52)与UNC-51样自噬激活激酶1 (UNC-51 like autophagy activating kinase 1, ULK1)复合物相结合,以及TAX1结合蛋白1 (TAX1-binding protein 1, TAX1BP1)与微管相关蛋白1轻链3 (microtubule-assoc...     

钙/钙调蛋白依赖性丝氨酸蛋白激酶的结构和功能

钙/钙调蛋白依赖性丝氨酸蛋白激酶(calcium/calmodulin-dependent serine protein kinase, CASK)属于膜相关鸟苷酸激酶（membrane associated guanylate kinase, MAGUK）家族.CASK具有多个不同蛋白质结合结构域，在细胞膜的特定区域，与其他蛋白质形成多种蛋白质复合体，参与组成细胞骨架.它通过衔接细胞外信号蛋白和细胞内骨架蛋白，协助功能蛋白质的转运和定位，以及细胞内的信号传递.此外CASK还可以进入细胞核影响基因转录调控，以及作用在神经突触膜上参与神经递质的释放.     

126Gln是人白细胞介素-2与其受体γ亚基结合的残基

人白细胞介素-2(IL-2)的126Gln是一个保守氨基酸,将126Gln突变为Asp后,测定了这一突变体与白细胞介素-2受体(IL-2R)不同亚基组合的结合能力,结果表明,突变体 126Asp-IL-2与 IL-2Rα βγ复合体可表现出较高的亲和力,与 IL-2 α,β复合体亲和力正常,而与 IL-2Rβγ复合体不具备亲和力.由此证实126Gln是人 IL-2与 IL-2Rγ亚基结合的残基.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

Modification of isolated subunit c of the F1F0-ATPase from Propionigenium modestum by dicyclohexylcarbodiimide

Subunit c of the F₁F₀-ATPase from Propionigenium modestum was extracted from the particulate cell fraction with chloroform/methanol. The protein was further purified by carboxymethyl cellulose chromatography and anion exchange HPLC in the organic solvent. SDS-PAGE of the purified protein indicated a single stained protein band migrating as expected for the c-subunit. Incubation of isolated subunit c in chlorform/methanol or aqueous buffer containing dodecyl-β- -maltoside with [¹⁴C]dicyclohexylcarbodiimide (DCCD) resulted in the incorporation of radioactivity into the protein. The rate of this reaction depended on the external pH; it was significantly faster in the more acidic than in the alkaline pH range. In the presence of Na⁺ subunit c was partially protected from labeling with [¹⁴C]DCCD at pH 6.1 and at pH 7.5, whereas no protection was evident at pH 5.5. At pH 7.5, the rate of subunit c labeling by [¹⁴C]DCCD in the presence of 20 mM NaCl was about 50% lower than in the absence of Na⁺ ions. The isolated c-subunit therefore apparently retains in part the Na⁺ binding site which, when occupied, diminishes the reactivity of the protein towards DCCD.     

Asp133 Residue in NhaA Na+/H+ Antiporter Is Required for Stability Cation Binding and Transport

Na⁺/H⁺ antiporters have a crucial role in pH and Na⁺ homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced. Asp133 and Lys 300 have been ascribed essential roles in this balance and, more generally, in the structure and function of the antiporter. In this work, we show the multiple roles of Asp133 in NhaA: (i) The residue's negative charge is critical for the stability of the NhaA structure. (ii) Its main chain is part of the active site. (iii) Its side chain functions as an alkaline-pH-dependent gate, changing the protein's conformation from an inward-facing conformation at acidic pH to an outward-open conformation at alkaline pH, opening the periplasm funnel. On the basis of the experimental data, we propose a tentative mechanism integrating the structural and functional roles of Asp133.     

梭梭Na+/H+逆向转运蛋白基因克隆及分析

采用RT-PCR、RACE方法从超旱生、耐盐植物梭梭中扩增出Na+/H+逆向转运蛋白基因的开放阅读框架,其核苷酸序列长1 683bp,推测的氨基酸序列全长为560个氨基酸残基。含有多个物种Na+/H+逆向转运蛋白基因的高度保守序列氨氯砒嗪脒的结合位点(LFFIYLIPPI)。序列一致性分析结果显示,该cDNA片段与同科植物NHX基因的一致性为70%～80%,但与不同科植物的一致性较低,仅为60%,表明该基因在进化上存在多样性,但它们都具有氨氯砒嗪脒结合位点,对Na+具有高度专一性,对植物的耐盐性起着重要作用。     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Early Signals in Serum-Induced Increases in Ouabain-Sensitive Na+-K+ Pump Activity and in Glucose Transport in Rat Skeletal Muscle Are Amiloride-Sensitive

Abstract: The acute effects of serum on sodium-potassium (Na⁺-K⁺) pump activity and glucose uptake in cultured rat skeletal muscle were studied. Addition of serum to myo-tubes in phosphate-buffered saline caused Na⁺-K⁺ pump activity (as measured by changes in the ouabain-sensitive component of both membrane potential and ⁸⁶Rb uptake) to increase, with peak effects obtained after 30 min. The effect was blocked completely by treatment with amiloride, but not by tetrodotoxin, which blocks voltage-dependent Na⁺ channels. On transfer of myotubes to Na⁺-free, choline buffer, resting Na⁺-K⁺ pump activity decreased to about 10% of that in phosphate-buffered saline. Addition of regular serum, but not Na⁺-free serum, caused Na⁺-K⁺ pump activity to increase slightly. Similar results were obtained with serum on glucose uptake, the peak effect being reached within 15 min. Stimulation of glucose uptake by serum was partially reduced by amiloride and was not altered by tetrodotoxin. Removal of external Na⁺ also eliminated serum effects on glucose uptake. The results demonstrate that there are similar signals involving Na⁺-H⁺ exchange for serum-induced increases in Na⁺-K⁺ pump activity and glucose transport. The lack of complete blockade of serum-induced elevation of glucose transport suggests an additional, as yet undefined, intracellular signal for stimulation of this transport system.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

长苞香蒲对人工盐碱湿地Na+和K+的吸收与转运特征

为揭示长苞香蒲(Typha domingensis)对盐生湿地生态系统中Na⁺和K⁺的吸收与转运特征,探讨长苞香蒲对盐生湿地的生态修复效果,该研究采用人工模拟盐生湿地的方法,设置CK(对照)、T1(浇灌100 mmol·L^-1盐水)、T2(浇灌200 mmol·L^-1盐水)及T3(浇灌300 mmol·L^-1盐水)4种不同盐浓度的人工湿地生态系统,并分别于5月5日(开始盐胁迫处理,S0)、5月30日(S1)、6月30日(S2)和7月30日(S3)测量其株高和干重、植株地上与地下部分Na⁺和K⁺的含量以及底泥和水体中Na⁺和K⁺的含量以分析长苞香蒲对盐碱湿地的脱盐作用。结果表明：(1)各处理的长苞香蒲的株高和干重随着处理时间的延长呈增加趋势,但与CK相比,各处理生长量随盐浓度升高出现下降趋势。(2)高浓度盐处理(T3)使长苞香蒲的地上部分和地下部分的Na⁺分别增加了2.5...