玉米根ABA结合蛋白的亚细胞定位及动力学性质

以玉米（Ｚｅａ ｍａｙｓＬ．）根或胚芽鞘为材料,经匀浆、分级离心得到胞质部分和膜部分（微粒体）,进一步用６．２％ （Ｗ／Ｗ ） Ｄｅｘｔｒａｎ Ｔ５００ 和ＰＥＧ ３３５０ 两相系统制备质膜,用１％ 和８％ （Ｗ ／Ｗ） Ｄｅｘｔｒａｎ Ｔ７０ 梯度离心制备液泡膜． 电镜鉴定及多种标志酶检测表明,制备获得了高纯度正向型质膜和富含液泡膜的组分,其它内膜的污染很少． 用微量放射配体结合（ＭＲＬＢ）实验证明,玉米根微粒体的ＡＢＡ专一性结合位点主要分布在液泡膜和质膜上,这两种膜组分与ＡＢＡ 的特异结合活性分别为２４８５．４ ｆｍ ｏｌ／ｍ ｇ ｐｒｏｔｅｉｎ 和１２５７．３ ｆｍ ｏｌ／ｍ ｇ ｐｒｏ－ｔｅｉｎ,玉米根段胞质部分结合活性最低（差一个数量级）．质膜上ＡＢＡ－ＢＰ与ＡＢＡ 的结合平衡解离常数（ＫＤ）为１．５７ ｎｍ ｏｌ／Ｌ．     

玉米根微粒体ABA结合蛋白的性质及逆境胁迫的影响

以玉米根微粒体为材料进行的微量放射配体结合（ＭＲＬＢ）实验表明玉米根微粒体膜上存在着ＡＢＡ结合位点,ＡＢＡ与ＡＢＡ结合蛋白（ＡＢＡ－ＢＰ）结合的最适ｐＨ为６．５,结合反应对温度（０℃和２５℃）不太敏感,ＡＢＡ与ＡＢＡ－ＢＰ的结合反应是一个动态平衡过程,５ｍｉｎ即可达最大结合（Ｂｍａｘ）的５０％,３０ｍｉｎ达到最大结合,１ｈ内基本保持不变。胰蛋白酶处理表明此结合位点为蛋白质,冻融实验则表明此蛋白与ＡＢＡ的结合不仅要求其自身具有特定构象而且需要有一定的膜脂环境,ＤＴＴ处理实验结果显示ＡＢＡ－ＢＰ中可能存在着二硫键。逆境处理可以提高玉米根微粒体膜对ＡＢＡ的结合活性,盐胁迫、渗透胁迫、干旱胁迫和热冲激处理分别使结合活性上升３４．９％、１７．８％、２３．１％和１３．３％。     

RBPMS不同剪接体的真核表达和亚细胞定位

目的：构建具有多种剪接形式的RNA结合蛋白（RBPMS）基因的真核表达载体,并在真核细胞中表达,确定不同形式的RBPMS在细胞中的定位。方法：采用PCR技术从人卵巢cDNA文库中扩增RBPMS基因的几种完整编码序列（命名为RBPl～RBP4）,克隆到带绿色荧光蛋白标签的pEGFP-C1表达载体上,转染人胚肾细胞293T,Western印迹鉴定RBPMS的表达,并利用激光共聚焦显微镜观察RBPMS不同剪接体在细胞中的定位。结果：限制性内切酶分析和DNA序列测定表明构建的重组表达载体正确,Western印迹实验证明RBP1～RBP4表达成功。通过激光共聚焦显微镜观察,RBP1／4围绕胞核在核膜的周围呈聚集状分布;RBP2则在细胞质和细胞核中均有分布,但会出现斑点状聚集;RBP3呈半月状紧密分布在细胞核周围;RBPMS中的RNA识别基序缺失后,这种现象消失,与空载体对照类似,在细胞核和细胞质中均有分布。结论：构建并表达了RBPMS基因的真核表达载体,RBPMS不同剪接体及RNA识别基序缺失后具有不同的亚细胞分布模式,提示具有不同的功能。     

人N-ras癌基因细胞型特异DNA结合蛋白

本文利用Southwestern印迹技术发现人肿瘤HT1080细胞染色质蛋白中一组与N-ras基因结合的蛋白,分子量约为150,105,95,90KDa,而与Ha-ras基因结合的一组蛋白,分子量约为160,115,100,55KDa,其中150KDa蛋白是N-ras基因特异的DNA结合蛋白,具有细胞型特异性,在HT1080细胞中含量最多,T24细胞次之,而在人HeLa细胞,淋巴细胞、肠细胞以及未转化的NTH3T3细胞中未被发现。此种蛋白可能与N-ras基因在HT1080细胞内的激活有密切关系。     

发育中玉米胚结合蛋白(BiP)的动态变化及定位

Western blot检测表明,在玉米胚发育过程中结合蛋白(BiP)含量与胚可溶性蛋白含量变化一致,在授粉16d后BiP含量随发育而增加;对热激不敏感.组织化学免疫定位表明,在玉米胚发育的不同时期,BiP主要定位在胚芽端、初生维管组织和糊粉层中,提示胚在构建器官的同时,也为其功能执行准备了条件;热激不影响其定位.     

Subcellular Localization of Rat Brain Insulin Binding Sites

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, gamma-aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mitochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r = 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.     

ABA对玉米响应干旱胁迫的调控机制

玉米(Zea mays L.)在生长期常常受到干旱的胁迫,而脱落酸(ABA)调节的生长响应是植物对逆境信号的一个基本反应. 本文对近年来国内外有关ABA提高玉米抗氧化防护系统,保护细胞免受氧化损伤,增加可溶性渗透剂(如脯氨酸)维持细胞内的水分,以及与其它激素相互作用影响玉米器官发育等的研究进展进行综述,以了解ABA调控玉米根系、叶片和籽粒的耐旱机理.     

Changes in Kinetic Characteristics of the ABA Binding Protein(s) of the Grapevine Fruit During Its Developmental Process

By using the micro-volume radio-ligand binding essay (MRLB), the changes in the kinetic characteristics of the ABA binding protein (s) of the Kyoho grapevine (V/t/s vinifera × V. labrusca) fruits during the different stages of fruit development have been studied. The changes in the berry volume growth, concentration of sugar, organic acids and ABA in fruit mesocarp have been determined especially for the studies of ABA-binding protein. The dissociation constant (Kd) and ABA-binding maximum volumes (Bronx)were determined by the Scatchard plots for the ABA-binding protein (s) in microsomes of the fruits. They were: Kd =17.5, 50. 0, 6.3, 13.3 nmol/L: Bmax=98.6, 523.0, 41.6, 85. 4 pmol/g protein respectively for the fruit developmental phase Ⅰ , Ⅱ, veraison and phase Ⅲ. The Scatchard plots showed a rectilinear function for all of the developmental phases including veraison, which suggests in the fruit microsomes at the same developmental phase, the existence of either one kind of the same or several different kinds of ABA-binding sites of identical or similar kinetic characteristics and of high affinity. In different developmental stages, however, changes of the protein at the ABA-binding sites might occur. The binding affinity of ABA-binding protein (s) for ABA was showed to be higher at veraison stage than in other developmental phases and this binding affinity increased nearly by 10 times from the phase Ⅱ to veraison, whereas the concentration (Bmax) of the ABA-binding protein (s) decreased to the minimum at veraison. The reason why such low concentration of ABA at veraison is capable to trigger the onset of fruit ripening might be due to the increase of the affinity of ABA-binding protein (s) for ABA at this time. The possible functions of the ABA-binding protein (s) for fruit development during the different developmental stages were discussed, and it is suggested that the protein (s) detected could be the putative receptor (s) or carrier (s) for the action of this plant hormone in grapevine.     

光亲和标记鉴定玉米根脱落酸结合蛋白

光亲和标记鉴定玉米根脱落酸结合蛋白吴忠义,陈珈,朱美君（北京农业大学生物学院,１０００９４）关键词结合蛋白;光亲和标记;ＡＢＡ;受体;微粒体脱落酸（ＡＢＡ）作为一大类植物激素,在高等植物的生长发育以及对逆境的适应过程中发挥着重要作用。在探讨激素作用的...     

Isolation and Characterization of Two Fatty Acid Binding Proteins from Mouse Brain

Abstract: Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.02 and 0.5 µ M , respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.01 and 0.7 µ M , respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. [³H]Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1 and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.     

Isolation and Purification of ABA Binding Protein from Abaxial Epiderm of Vicia faba Leaf

Abscisic acid (ABA) was efficiently cross-linked to Sepharose 4B (6 ~8 mmol ABA/L gel) by an ann of 10-atom carbon chain. Solubilized ABA-BP (ABA binding protein) was allowed to bind to the gel, while unrelated proteins were removed by washing with a gradient of NaC1 buffer. The ABA-BP was eluted with 1 mmol/L ABA. Since ABA at high concemration can interfere with both the binding activity assay and protein analysis, the fractions eluted with ABA were passed through a Sephadex G-25 column to remove the ABA. Fractions containing the binding activity were pooled, concentrated with uhm-fihration. The maximum binding capacity (BMAX) of the purified ABA-BP was 58.33 nmol/g protein, and the Kd was 21 nmol/L, with an approximately 112 folds increase of purity. SDS-PAGE identification of the purified ABA-BP revealed a major protein band with a molecular weight of about 44.2 kD, and a purity of approximately 90 %.     

Characterization and Subcellular Localization of l-[3H]Carnitine Binding Sites in Rat Brain

Abstract: In the present study, we investigated the existence of a binding site for l -carnitine in the rat brain. In crude synaptic membranes, l -[³H]carnitine bound with relatively high affinity (K_D = 281 nM) and in a saturable manner to a finite number (apparent B_max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K_off of 0.018 min^?1 and a K_on of 0.187 × 10^?3 min^?1 nM^?1. Binding was highest in spinal cord, followed by medulla oblongata-pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l -[³H]Carnitine binding was stereoselective for the l -isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l -[³H]carnitine binding was l -carnitine followed by propionyl-l -carnitine. Acetyl-l -carnitine and isobutyryl-l -carnitine showed an affinity ～500-fold lower than that obtained for l -carnitine. The precursor γ-butyrobetaine had negligible activity at 0.1 mM. l -Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl -carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l -carnitine membrane binding sites whereas l -carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l -[³H]carnitine binding site should be essentially presynaptic.     

钙结合蛋白CIB1在293T细胞中的表达及亚细胞定位研究

分析和比对钙结合蛋白CIB1在人类、大鼠、小鼠中氨基酸序列差异,并研究CIB1蛋白在293T细胞中的表达及亚细胞定位情况。通过Western Blot分析发现293T细胞本身几乎检测不到CIB1的表达。进一步采用CIB1外源性质粒转染,通过免疫荧光分析实验,在激光共聚焦显微镜下观察转染后不同时间293T细胞中CIB1的表达情况及亚细胞定位变化。实验结果表明,随着转染时间的增加,CIB1在293T细胞中的表达有逐渐增强的趋势,并且发生从细胞核向细胞质的转位现象。该实验结果对了解CIB1亚细胞定位变化及功能研究具有重要参考价值。     

葡萄果实发育过程中脱落酸结合蛋白动力学特性的变化

联系葡萄果实发育不同阶段生长动态、果肉组织中糖、酸和ＡＢＡ 含量变化的测定, 利用微量放射配基结合法,通过Ｓｃａｔｃｈａｒｄ 作图,分析了巨峰葡萄（Ｖｉｔｉｓｖｉｎｉｆｅｒａ×Ｖ． ｌａｂｒｕｓｃａ）果实生长发育第Ⅰ期、第Ⅱ期、始熟期和第Ⅲ期膜联系的ＡＢＡ 结合蛋白的动力学特性参数,其解离常数（Ｋｄ） 依次分别为１７．５、５０．０、６．３、１３．３ ｎｍ ｏｌ／Ｌ;最大结合容量依次分别为９８．６、５２３．０、４１．６、８５．４ ｐｍ ｏｌ／ｇ 蛋白。这４ 个时期的Ｓｃａｔｃｈａｒｄ 图都是一条直线, 说明在同一发育时期,果实微粒体上可能存在一种相同的或若干种不同的但动力学特性相同或相似的高亲和力的ＡＢＡ 结合位点。ＡＢＡ 结合蛋白与ＡＢＡ 的亲和力在始熟期高于其它时期,特别是从第Ⅱ期到始熟期,亲和力提高了将近１０ 倍,而其浓度却在始熟期降到最低。始熟期果实组织中低浓度的ＡＢＡ 启动成熟的原因可能是由于ＡＢＡ 结合蛋白与ＡＢＡ 亲和力的提高。讨论了ＡＢＡ 结合蛋白在果实发育不同时期的功能,推测这种（些）蛋白可能是ＡＢＡ 作用的受体或载体     

两个拟南芥未知功能蛋白的亚细胞定位研究

蛋白质的亚细胞定位对于深入了解该蛋白质所行使的生理功能具有重要意义。经生物信息学预测,两个拟南芥未知功能基因At4g16410与Atl gI8060编码蛋白含有叶绿体定位信息。我们分别克隆了这两个基因5’端长199bp与220bp的DNA片段,与绿色荧光蛋白（GFP）基因构建重组表达载体pMON530-cTP1-GFP与pMON530-cTP2-GFP,经农杆菌介导转化拟南芥。两种转基因植株经激光共聚焦显微镜观察,GFP荧光仅在叶绿体中观察到,表明所克隆的两段DNA序列编码的多肽能够将At4gl6410与Atlgl8060编码蛋白质引导进入叶绿体,确定这两个蛋白质均为叶绿体蛋白质。     

拟南芥ABA不敏感型突变体及其ABA结合特性

拟南芥的脱落醚（ABA）不敏感型突变体abi2,在对ABA的敏感性、气孔开度及种子休眠方面,与野生型有明显差异。通过3H－ABA与野生型对的亚细胞组分的结合分析,表明38000×g组分特异结合活性最高,结合最适温度为20℃,最适保温时间：20℃时为70min;0℃时为90min。由饱和曲线的Scatchard分析表明：abi2存在一种ABA结合位点,野生型有两种ABA结合位点。对3H－ABA结合的38000×g组分的SDS－PAGE电泳分析表明：野生型有3个结合活性峰,而abi2只有1个结合活性峰。     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Microextraction of Nuclear Proteins from Single Maize Embryos

The analysis of DNA binding proteins can be difficult when only small quantities of tissue expressing the desired protein are available. We present a protocol for the preparation of nuclear extracts from as little as 100 mg of tissue. This protocol is well suited for extraction of DNA binding proteins from tissues that are difficult to obtain in large quantities such as maize embryos.