亲和层析法分离大鼠脂肪细胞膜上腺苷A1受体

本文采用腺苷亲和层析法从大鼠脂肪细胞膜上分离出了一种亚基分子量为３８ｋＤ的腺苷结合蛋白质。此蛋白在ＳＤＳ－聚丙烯酰胺凝胶电泳上显示单一带,糖蛋白染色阳性;能与〔８－＾３Ｈ〕腺苷特异结合（Ｋｄ＝０．２６９ｎｍｏｌ／Ｌ,Ｂｍａｘ＝６．０５ｐｍｏｌ／ｍｇ．Ｐｒ）;结合抑制实验表明它与腺苷Ａ１受体激动剂Ｒ－ＰＩＡ,Ａ２受体激动剂ＮＥＣＡ和腺苷的亲和力大小顺序为：Ｒ－ＰＩＡ＞腺苷＞ＮＥＣＡ。这表明所分离出的     

猪肝微粒体NADH—细胞色素b5还原酶的纯化及特性分析

采用硫酸铵分级分离,Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤,ＤＥＡＥ－纤维素离子交换层析以及５＇－ＡＭＰ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析,从猪肝微粒体中纯化得到可溶性的ＮＡＤＨ－细胞色素ｂ５还原酶,提纯倍数为７５０－８００。总回收率为４０％左右。纯化的酶呈典型的黄素蛋白吸收光谱,Ａ２７３／Ａ４６０比值为５．８．在ＳＤＳ－聚丙烯酰胺凝胶电泳板上呈单一的蛋白质区带,分子量为３２ｋｄ。ＮＡＤＨ和２,６－     

血链球菌拮抗可疑牙周病原菌机理探讨：Ⅱ细胞素的产生及…

血链球菌产生细菌素至培养上清液中,无论是需氧或厌氧环境,血链球菌均可产生血逻素。采用无水乙醇沉淀法提取血链球菌培养上清液中的蛋白质,运用ＳＤＳ－ＰＡＥＧ分析其蛋白成份,发现１３０ｋｄ、１２０ｋｄ、７４ｋｄ、和５６ｋｄ四条蛋白区带,通过电泳凝胶弥散法抑菌试验显示,５６ｋｄ蛋白带具很强的抑菌活性,提示此蛋白质可能为血链素的所在位置。     

血链球菌拮抗可疑牙周病原菌机理探讨Ⅱ细菌素的产生及影响因素

血链球菌产生细菌素（血链素）至培养上清液中，无论是需氧或厌氧环境，血链球菌均可产生血链素。采用无水乙醇沉淀法提取血链球菌培养上清液中的蛋白质，运用ＳＤＳ－ＰＡＧＥ分析其蛋白成份，发现有１３０ｋｄ、１２０ｋｄ、７４ｋｄ、和５６ｋｄ四条蛋白区带，通过电泳凝胶弥散法抑菌试验显示，５６ｋｄ蛋白带具很强的抑菌活性，提示此蛋白质可能为血链素的所在位置。     

猪肝微粒体NADH－细胞色素b_5还原酶的纯化及特性分析

采用硫酸铵分级分离,ＳｅｐｈａｄｅｘＧ－１００凝胶过滤,ＤＥＡＥ－纤维素离子交换层析以及５＇－ＡＭＰ～Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,从猪肝微粒体中纯化得到可溶性的ＮＡＤＨ－细胞色素ｂ５还原酶,提纯倍数为７５０～８００。总回收率为４０％左右。纯化的酶是典型的黄素蛋白吸收光谱,Ａ２７３／Ａ４６０比值为５．８。在ＳＤＳ－聚丙烯酸胺凝胶电泳板上呈单一的蛋白质区带,分子量为３２ｋｄ。ＮＡＤＨ和２,６－二氯酚靛酚的Ｋｍ值分别为２４和４５μｍｏｌ／Ｌ,以２,６－二氯酚靛酚为底物时,该还原酶的催化作用可能为乒乓机制。该酶的纯化为分子水平研究其反应机制及制备相应的抗体以建立免疫学检测方法创造了条件。     

丝瓜籽中蛋白质生物合成抑制蛋白的分离,纯化及性…

丝瓜种籽,经捣碎,抽提,硫酸铵分级沉淀,ＣＭ－５２离子交换层析,Ｓｅｐｈａｃｒｙｌ Ｓ－１００分子筛,阳离子交换ＦＰＬＣ等步骤,分离到两种单链蛋白质生物合成抑制蛋白：Ｌｕｆｆｉｎ－Ａ和Ｌｕｆｆｉｎ－Ｂ。它们都是等电点接近１０的碱性蛋白,ＳＤＳ－ＰＡＧＥ测定分子量分别约为２７ｋｄ和２８ｋｄ,氨基酸组成分析表明两者具有很大同源性,但免疫双扩散及ＥＬＩＳＡ检测证明两者的免疫原性有差异。Ｌｕｆｆｉｎｓ对兔     

天花粉同工凝集素－1双链的拆分和鉴定

天花粉同工凝集素－１经巯基乙醇还原,碘代乙酰胺保护,其链间二硫键被打开,但仍非共价结合在一起。我们利用尿素变性的Ｑ－Ｓｅｐｈａｒｏｓｅ离子交换层忻分离了此凝集素的两条链。氨基酸组成测定与其他３种肽链作一比较,它们都含有较多的酸性和羟基氨基酸。蛋白质印迹显示ＴＫＬ的抗血清不仅能与ＴＫＬ－１的两条链分别反应,也能与天花粉毒蛋白及蓖麻毒蛋白的Ａ链起作用。溴化氰裂解的ＳＤＳ－ＰＡＧＥＲ肽谱表明天花粉凝集素的两条链与天花粉毒蛋白含有类似的裂解片段,在分子量１６ｋｄ左右有相同的电泳条带。ＴＫＬ－１两亚基的Ｎ末端序列已经测定,同源性比较发现其３３ｋｄ亚基的Ｎ末端序列与天花粉毒蛋白、蓖麻毒蛋白的一些肽段类似。迄今已有的证据表明ＴＫＬ与ＴＣＳ等是一些非常相关的蛋白质。     

Epstein─Barr病毒膜抗原在中国仓鼠卵巢（CHO）细胞中的表达

将切去３’端穿膜序列的ＥＢ病毒膜抗原（ＭＡ）基因,插入ｐＳＶ２－ｄｈｆｒ质粒的ＳＶ４０早期启动子下游,构建了真核表达载体ｐＳＶ２－ｄｈｆｒＧＰＴＲ,使两个ＳＶ４０早期启动子分别调控ＭＡ和二氢叶酸还原酶（ｄｈｆｒ）基因。将该重组质粒转化ＣＨＯ－ｄｈｆｒ细胞,在选择培养基中筛选阳性克隆,用氨甲喋呤加压扩增,建立了表达ＥＢＶ－ＭＡ的克隆细胞系。ｗｅｓｔｅｒｎｂｌｏｔ分析证明,所表达的蛋白的分子量大约为３４０ｋｄ和２２０ｋｄ。经过细Ｓｅｐｈａｒｏｓｅ２Ｂ琼脂糖凝胶层析初步纯化的抗原与福氏佐剂混合免疫小鼠,２周后小鼠血清中出现明显的ｇｐ３４０／２２０特异性抗体,表明切去嵌膜区结构的ＥＢＶ－ＭＡ基因在ＣＨＯ细胞中的表达产物具有同天然膜抗原相似的分子量大小、糖基化程度、免疫特异性和免疫原性,可望成为ＥＢ病毒人用基因工程亚单位疫苗。     

ＮＳＦ在细胞分泌中的作用

Ｎ－乙基顺丁烯二酰亚胺敏感性的融合蛋白（Ｎ-ethylmaleimide-sensitive fusion protein,NSF)是在细胞内膜泡转运中发挥重要作用的一种ＡＴＰ酶。它在进化上高度保守。参与了不同物种、不同细胞类型的分泌过程。ＮＳＦ的作用需要可溶性的ＮＳＦ附着蛋白（ＳＮＡＰ）和ＳＮＡＰ受体（ＳＮＡＲＥ）的辅助。“ＳＮＡＲＥ”假说认为：ＮＳＦ主要在分泌小泡与靶膜融合时起作用。但近年来倾向于认为其在膜融合过程的多个阶段均发挥作用。     

高分子量麦谷蛋白１４和１５亚基的纯化、Ｎ—末端序列及部分生化特性研究

用ＳＤＳ－ＰＡＧＥ制备电泳技术结合一种新的凝胶中蛋白质显色方法,对普通小麦（Triticum aestivum)小偃六号的高分子量麦谷蛋白１４和１５亚基进行了有效的分离纯化,将其转印于ＰＶＤＦ膜上测定了Ｎ－端的氨基酸顺序,通过比较了发现它们与已知序列的其他的高分子是麦谷蛋白亚基高度同源。用两种双向电泳技术确定了它们的等电点（ＰＩ）属于碱性范围。     

Characterization of Membrane-Bound and Solubilized High-Affinity Binding Sites for 5'-N-Ethylcarboxamido[3H]adenosine from Bovine Cerebral Cortex

Abstract: A high-affinity binding site for 5'- N -ethylcarboxamido[³H]adenosine ([³H]NECA) from bovine cerebral cortex has been characterized in its membrane-bound and solubilized state after gel filtration on Sepharose CL-6B. For detection of this site in membranes, it was necessary to remove metabolites with high affinities for this site enzymatically, e.g., adenosine by addition of adenosine deaminase and inosine by addition of nucleoside phosphorylase. The pore-forming peptide antibiotic alamethicin further enhanced binding of [³H]NECA to this site in membranes. In contrast to adenosine receptors and the adenotin-like low-affinity binding protein, this novel site was extremely sensitive against treatment with the sulfhydryl alkylating agent N -ethylmaleimide. In competition experiments, this site could be differentiated from adenosine receptors by its high affinity for adenine nucleotides and its lack of affinity for adenosine receptor antagonists. Inosine and its derivative S -(4-nitrobenzyl)-6-thioinosine were relatively potent ligands with K _i values in the high nano- and low micromolar range, respectively. We conclude that the high-affinity NECA binding site described previously in bovine striatum is not exclusively located in the striatum, but can also be detected in membrane preparations and soluble extracts of bovine brain cortex.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A₁ adenosine receptor (A₁R) is a second ecto-ADA binding protein. Binding of ADA to A₁R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A₁R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A₁R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A₁R.     

Purification and characterization of the D2 cell adhesion protein: Analysis of the postnatally regulated polymorphic forms and their cellular distribution

The developmentally regulated, D2 cell adhesion protein has been purified from 10–12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5–1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (>200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.     

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: Characterization by radioligand binding and photoaffinity labelling

A₁ adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[³H]dipropyl-xanthine ([³H]DPCPX) gave a K_d value of 0.7 nM and a B_max value of 82 ± 13 fmol/mg protein. For the highly A₁-selective agonist 2-chloro-N⁶-[³H]cyclopentyladenosine ([³H]CCPA) a K_d value of 1.7 nM and a B_max value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [³H]DPCPX binding gave a pharmacological profile with R-N⁶-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5′-N-ethylcarboxamido-adenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine ([¹²⁵I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A₁ receptors labelled in brain membranes. Therefore, coated vesicles contain A₁ adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A₁ receptors in these vesicles are not a processed receptor form. These results confirm that A₁ receptors in coated vesicles are coupled to a G-protein, and it appears that the A₁ receptor systems in coated vesicles and in plasma membranes are identical.     

Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor

An approximate 140-fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A1 adenosine receptor antagonist. As assessed by [3H]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol/mg protein. Interaction of solubilized A1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3H]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 +/- 22 pmol/mg protein with typically 5-15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R-PIA greater than S-PIA greater than NECA. In purified preparations, the photoaffinity probe [125I]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38,000 which has previously been shown to be the A1 adenosine receptor binding subunit.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

亲和层析法分离大鼠脂肪细胞膜上腺苷A_1受体

本文采用腺苷亲和层析法从大鼠脂肪细胞膜上分离出了一种亚基分子量为38kD的腺苷结合蛋白质。此蛋白在SDS-聚丙烯酰胺凝胶电泳上显示单一带,糖蛋白染色阳性;能与[8-~3H]腺苷特异结合(Kd=0.269nmol/L,Bmax=6.05pmol/mg.Pr);结合抑制实验表明它与腺苷A_1受体激动剂R-PIA、A_2受体激动剂NECA和腺苷的亲和力大小顺序为:R-PIA>腺苷>NECA。这表明所分离出的38kD蛋白是大鼠脂肪细胞膜上的腺苷A_1受体。     

Characterization of the Solubilized A1 Adenosine Receptor from Rat Brain Membranes

A1 adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A1 adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-N6-[3H]phenylisopropyladenosine([3H]PIA) with KD values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A1 adenosine receptors could be labelled not only with the agonist [3H]PIA but also with the antagonist 1,3-diethyl-8-[3H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein Ni and that all regulatory functions are retained on solubilization.     

Fibulin, a novel protein that interacts with the fibronectin receptor beta subunit cytoplasmic domain

A 100 kd protein was isolated from tissue and cell extracts by affinity chromatography on a synthetic peptide representing the cytoplasmic domain of the fibronectin receptor beta subunit. The 100 kd protein also bound to native fibronectin receptor, and this binding could be reversed with EDTA. Calcium may be the divalent cation required for the binding since the 100 kd protein was found to bind 45Ca2+. The N-terminal amino acid sequence of the 100 kd protein was not similar to any sequence in a protein data base. Immunofluorescent staining of cells cultured on fibronectin showed the 100 kd protein coinciding with the fibronectin receptor beta subunit in sites of substrate contact. Therefore this protein, which we term fibulin, interacts with the fibronectin receptor in vitro and associates with the receptor in vivo. Fibulin is a potential mediator of interactions between adhesion receptors and the cytoskeleton.