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     

腺苷转运体亲和层析分离

本文合成了一种腺苷亲和层析凝胶,并采用亲和层析法从牛脑细胞膜上分离出了几种膜上结合的腺苷结合蛋白质。这些蛋白质在ＳＤＳ－ＰＡＧＥ电泳凝胶上为单一或主要的蛋白带,分子量分别为６４ｋｄ,４５ｋｄ,３５ｋｄ。腺苷转运体抑制剂潘生丁和ＮＢＭＰＲ对６４ｋｄ蛋白与＾３ｈ－腺苷的结合抑制作用远强于腺苷受体的激动剂ＮＥＣＡ和Ｒ－ＰＩＡ;这表明６４ｋｄ蛋白为牛脑细胞膜上结合的腺苷转运体。     

缓激肽受体B1R对SD大鼠阴茎勃起功能影响的实验研究

摘要 目的：研究缓激肽受体B1R对大鼠阴茎勃起功能的影响。方法：通过腹腔注射B1受体激动剂[Des-Arg⁹]-Bradykinin与B1受体抑制剂Lys-(des-Arg⁹, Leu⁸)-Bradykinin,观察各组大鼠阴茎勃起功能,通过HE染色和Masson染色观察大鼠阴茎组织形态变化及纤维化水平的变化,通过Western-blot 检测大鼠阴茎组织TGF-β1、TNF-α与IL-6等炎症因子的表达情况。结果：（1）B1受体激动剂显著抑制大鼠阴茎勃起功能,并升高阴茎胶原纤维/肌原纤维比值;而B1受体抑制剂显著提升大鼠阴茎勃起功能,并降低阴茎胶原纤维/肌原纤维比值;（2）B1受体激动剂显著升高大鼠阴茎TGF-β1、TNF-α与IL-6蛋白表达水平,而B1受体抑制剂降低大鼠阴茎TGF-β1、TNF-α与IL-6蛋白表达水平。结论：B1受体可能通过炎症因子、阴茎组织纤维化影响阴茎勃起功能。     

大鼠前体脂肪细胞分化过程中波形纤维形态结构的变化和波形纤维蛋白基因表达的研究

通过对不同分化阶段的大鼠前体脂肪细胞中波形纤维蛋白的结构形态和分布的间接免疫荧光观察,发现随着前体脂肪细胞的分化,波形纤维形态结构发生特异性改变,即从前期的围绕细胞核聚集且向细胞周边平行延伸到后期的围绕脂滴间隔形成致密笼状结构。此外,通过地高辛标记的寡核苷酸探针的原位杂交和免疫印迹研究了前体脂肪细胞的分化对波形纤维蛋白基因表达的影响。结果表明,分子量为57kD的波形纤维蛋白在mRNA水平和蛋白水平的表达贯穿于前体脂肪细胞分化的全过程,表达量呈递减趋势。这提示在前体脂肪细胞分化中,波形纤维与脂滴的特异性结合对于脂滴的前体脂肪细胞分化有着功能性的联系,特别是对脂肪细胞的脂滴形成极可能起到支撑的作用。     

RT-PCR克隆籼稻叶绿素a/b结合蛋白基因全长cDNA及序列的in silico分析

根据日本晴cab4基因序列(GenBank:AK104499.1)设计引物,用RT-PCR的方法从籼稻9311中克隆了叶绿素a/b结合蛋白基因的全长cDNA,命名为cab-9311(cab gene from 9311)。insilico分析表明:cab-9311与cab4基因同源性为99%,编码的蛋白含有244个氨基酸,与cab4基因编码的蛋白同源性为98%。蛋白分子质量为26.9kD,理论等电点为6.52。第54位～第216位氨基酸是一个典型的叶绿素a/b结合蛋白功能域(chlorophyll a/bbinding domain)。跨膜分析和蛋白质三级预测显示,该蛋白在C端有一个典型的跨膜区。亚细胞定位分析表明该蛋白定位于叶绿体,是一个叶绿体内囊体膜上的锚定蛋白。     

亲和层析法分离腺苷结合蛋白质

本文报告一种新的腺苷亲和层析凝胶的合成方法。利用这种凝胶可从大鼠心脏、肝脏及小牛主动脉平滑肌的水溶部份分离出几种腺苷结合蛋白质,其亚基分子量(据SDS-PAGE)分别为35,000、37,000、46,000、43,000及15,300Dal。现已证明,35,000Dal蛋白质是乳酸脱氢酶及苹果酸脱氢酶,43,000Dal蛋白质是腺苷激酶,46,000Dal蛋白质可能是S-腺苷同型半胱氨酸水解酶。15,000Dal蛋白质前人未有报道。它对腺苷具有高度特导性和亲和力,推测是腺苷的细胞内受体和/或载体。测定了这种低分子量腺苷结合蛋白质的氨基酸组成及某些物理常数:pI=6.5;沉降系数2.42S,微分比容0.727cm~3/g,与腺苷复合物的解离常数K_D=2.3μM。     

棕色固氮菌突变种UW3部分提纯的固氮酶CrFe蛋白溶液中残存细菌铁蛋白的晶体生长及鉴定

从无钼、无氨而含铬的固氮培养基中生长的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3中纯化得到了部分纯的CrFe蛋白。在试图培养CrFe蛋白大晶体时发现,棕色晶体和砖红色晶体可同时或单独出现。SDS-PAGE和厌氧天然PAGE皆表明,棕色晶体主要由与固氮酶钼铁蛋白(Av1)类似大小的亚基(～60kD)组成,而砖红色晶体则由～20kD亚基组成。免疫分析表明只有～60kD的亚基可与固氮酶钼铁蛋白的抗体反应,而～20kD亚基则无这种反应。在部分纯的CrFe蛋白溶液中,～20 kD的总蛋白含量远低于～60 kD蛋白的含量,表明由这种小亚基组成的蛋白只是CrFe蛋白溶液中的一种污染蛋白。用3,5-二氨基苯甲酸染色的天然电泳表明,形成砖红色和棕色晶体的蛋白是迁移率不同的两种含铁蛋白。质谱分析表明砖红色晶体蛋白为棕色固氮菌的细菌铁蛋白。分辨率为2.34的X射线衍射结果也表明,砖红色晶体属于H3空间群,晶胞参数为a=124.965,b=124.965和c=287.406。即将发表的三维结构解析表明,此砖红色晶体确为24聚体的细菌铁蛋白。     

腺苷A1受体在离体延髓脑片上对节律性呼吸的调制

实验旨在探讨腺苷A1受体在对基本呼吸节律调制中的可能作用。制作新生大鼠离体延髓脑片标本,主要包含面神经后核内侧区(themedial region of the nucleus retrofacialis,mNRF),并保留完整的舌下神经根。以改良Kreb‘s液灌流脑片,记录mNRF吸气神经元的电活动,并同步记录舌下神经根呼吸节律性放电(respiratory rhythmical discharge activity,RRDA)。在灌流液中先分别单独给予腺苷A1受体的特异性拮抗剂8-环戊-1,3-二丙基黄嘌呤(8-cyclopenty 1-1,3-dipropylxanthine,DPCPX)和特异性激动剂R-苯异丙基-腺苷(R-phenylisopropyl-adenosine,R-PIA);再分别先后给予R-PIA和R-PIA DPCPX,观察RRDA和吸气神经元电活动的变化。结果显示,给予腺苷A1受体拮抗剂DPCPX后,呼气时程和呼吸周期明显缩短,吸气神经元中期放电的频率和峰频率显著增大;给予腺苷Al受体激动剂R-PIA后,吸气时程、积分幅度和吸气神经元中期放电的频率和峰频率均显著降低,呼吸周期明显延长,且R-PIA的呼吸抑制作用可部分地被DPCPX逆转。实验结果提示,腺苷A1受体可能通过介导吸气神经元的抑制性突触输入参与节律性呼吸的调制。     

阿必鲁肽：一种超长效Ⅱ型糖尿病治疗新药

阿必鲁肽(Albiglutide)为新型GLP-1受体激动剂,可通过与胰岛β细胞表面的GLP-1受体结合,激活由细胞膜偶联G蛋白调控的胞内信号传导系统,修复及促进胰岛β细胞功能,增加胰岛素的分泌,从而降低血糖浓度及糖化血红蛋白(Hb A1c)水平,是治疗Ⅱ型糖尿病的理想药物。目前开发的各种长效GLP-1受体激动剂成为近二十年糖尿病药物的研究热点。本文对一种极具市场潜力的新型GLP-1受体激动剂阿必鲁肽(Albiglutide)的分子结构、作用机制、剂量研究、药代动力学、药效学以及副作用等临床试验研究进展进行综述。     

棕色固氮菌突变种UW3部分提纯的固氮酶CrFe蛋白溶液中残存细菌铁蛋白的晶体生长及鉴定

从无钼、无氨而含铬的固氮培养基中生长的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3中纯化得到了部分纯的CrFe蛋白.在试图培养CrFe蛋白大晶体时发现,棕色晶体和砖红色晶体可同时或单独出现.SDS-PAGE和厌氧天然PAGE皆表明,棕色晶体主要由与固氮酶钼铁蛋白(Av1)类似大小的亚基(～60 kD)组成,而砖红色晶体则由～20kD亚基组成.免疫分析表明只有～60kD的亚基可与固氮酶钼铁蛋白的抗体反应,而～20kD亚基则无这种反应.在部分纯的CrFe蛋白溶液中,～20 kD的总蛋白含量远低于～60 kD蛋白的含量,表明由这种小亚基组成的蛋白只是CrFe蛋白溶液中的一种污染蛋白.用3,5-二氨基苯甲酸染色的天然电泳表明,形成砖红色和棕色晶体的蛋白是迁移率不同的两种含铁蛋白.质谱分析表明砖红色晶体蛋白为棕色固氮菌的细菌铁蛋白.分辨率为2.34 A的X射线衍射结果也表明,砖红色晶体属于H3空间群,晶胞参数为a=124.965A,b=124.965A和c=287.406 A.即将发表的三维结构解析表明,此砖红色晶体确为24聚体的细菌铁蛋白.     

Biochemical and immunological characterization of A1 adenosine receptors purified from human brain membranes.

The A1 adenosine receptor was purified approximately 13,000-fold to apparent homogeneity from human cerebral cortex membranes using a novel affinity-chromatography system developed for the purification of rat brain and rat testis A1 adenosine receptors [Nakata, H. (1989) J. Biol. Chem. 264, 16,545-16,551; Nakata, H. (1990) J. Biol. Chem. 265, 671-677]. The purified human brain receptor showed the ligand-binding specificity expected of the A1 adenosine receptor. The Bmax and Kd for the purified receptor with a specific A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, were approximately 16 nmol/mg protein and 2 nM, respectively. SDS/PAGE of the purified receptor preparation showed one broad protein band of molecular mass of approximately 35 kDa, which is very similar to that of purified A1 adenosine receptor from rat brain membranes. Endoglycosidase F treatment of the purified receptor reduced the molecular mass to approximately 30 kDa, suggesting that the human brain A1 adenosine receptor is a glycoprotein. Comparison of the purified human and rat brain A1 adenosine receptors by peptide mapping after the proteolytic digestion showed minor differences between these receptors. Immunological comparisons of the human brain A1 adenosine receptor with rat brain A1 adenosine receptor using polyclonal antibodies against the purified rat brain A1 adenosine receptor showed that the antibodies react preferentially with the rat brain receptor and weakly with human brain receptor.     

A1 adenosine receptor of rat testis membranes. Purification and partial characterization

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.     

Sensitization by chronic diazepam treatment of A2A adenosine receptor-mediated relaxation in rat pulmonary artery

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.     

Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach

Monoclonal antiadenosine receptor antibodies have been raised by an auto-anti-idiotypic approach. BALB/c mice were immunized with adenosine 6-aminocaproyl-bovine serum albumin. Hybridoma cell lines were raised and lines that secreted antibodies that bound to rabbit antiadenosine antibodies were obtained. Two such monoclonal antibodies, AA18 and AA21, were studied in detail and found to be directed at adenosine receptors by the following criteria. They inhibited the binding of [3H] adenosine to rabbit antiadenosine antibodies that had binding characteristics similar to those of adenosine receptors. They bound to rat brain membranes and binding could be inhibited by N6-cyclohexyladenosine and L-N6-phenylisopropyladenosine, both adenosine receptor agonists. They also inhibited the binding of [3H]L-N6-phenylisopropyladenosine to rat brain membranes. In functional assays, they inhibited adenylate cyclase of rat brain membranes, but had no effect on adenylate cyclase of rat hepatic membranes, indicating that they mimic agonists of the A1 receptor, therefore, carrying an "internal image" of the adenosine molecule. When adenosine receptors of rat brain membranes were solubilized with 1% cholic acid, partially purified on an adenosine 6-aminocaproyl AH-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, both AA18 and AA21 recognized a 62,000 band under nonreducing conditions, and a major band of 36,000 under reducing conditions. We conclude that the auto-anti-idiotypic route has yielded specific antibodies that recognize the A1 adenosine receptor.     

Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins

A low-affinity adenosine binding protein has recently been distinguished from the adenosine A2 receptor and purified from human placental membranes. Soluble human placental extracts contain an adenosine binding activity that has properties similar to those of the membrane low-affinity adenosine binding protein. The binding protein was purified from soluble human placental extracts 134-fold to 89% purity with a Bmax of 2.5 nmol/mg. It comprises 0.7-0.9% of the soluble protein. The major purified soluble protein has a subunit molecular mass of 98 kDa and a Stokes radius identical with that of the membrane-bound adenosine binding protein. Competition analysis of the soluble protein revealed similar affinities and an identical potency order for displacement of 5'-(N-ethylcarbamoyl)[2,8-3H]adenosine ([3H]NECA) as follows: NECA greater than 2-chloroadenosine greater than adenosine greater than (R)-N6-(2-phenylisopropyl)adenosine. The soluble binding protein was more acidic than the membrane binding protein as revealed by a comparison of the elution properties during ion exchange chromatography. A second form of soluble adenosine binding activity comprised 17% of the major form and had a charge similar to that of the membrane binding protein, a smaller Stokes radius, and a subunit molecular mass of 74 kDa. Carbohydrate composition analysis revealed that the major soluble form has 4.3% carbohydrate by weight as compared to the membrane-associated form, which has 5.5% carbohydrate by weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Histidine Residues in the Adenosine A2a Receptor Ligand Binding Site

Abstract: The pH dependency of the binding of ligands to adenosine A_2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two- to fourfold increase in affinity was observed for CGS-21680, 5'- N -ethylcarboxamidoadenosine, adenosine, 3'-deoxyadenosine, 5'-deoxyadenosine, inosine, and N ⁶-methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2'-deoxyadenosine, although 2'-methoxyadenosine binding was pH dependent. This effect on the affinity of CGS-21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2'-hydroxyl group of adenosine agonists.     

Protein kinases associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates.

When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

Purification of A1 adenosine receptor from rat brain membranes

The A1 adenosine receptor from rat brain membranes has been purified about 50,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized xanthine amine congener-agarose, hydroxylapatite chromatography, and reaffinity chromatography. The overall yield starting from the membranes was approximately 4%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified preparation gave a broad single band of an apparent molecular weight of 34,000 either by silver staining or autoradiogram after radioiodination. The purified receptor bound approximately 24 nmol of 8-cyclopentyl-1,3-[3H]dipropylxanthine/mg of protein with a dissociation constant of 1.4 nM. This maximum specific binding value is consistent with the expected theoretical specific activity (29.4 nmol/mg) for a protein with a molecular mass of 34,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. Affinity-labeling experiments using [3H]p-phenylenediisothiocyanate-xanthine amine congener showed that the Mr = 34,000 protein band contained the ligand-binding sites. The purified receptor gave a typical A1 adenosine receptor pharmacological specificity similar to that of unpurified receptor preparations.     

Alkylxanthines as adenosine receptor antagonists and membrane phosphodiesterase inhibitors in central nervous tissue: evaluation of structure-activity relationships

A series of alkylxanthines were examined as antagonists of the adenosine A₁-receptor in rat brain synaptosomal membranes and as inhibitors of brain membrane phosphodiesterase. The structure-activity relationships showed that the addition of certain substituting groups on the 8th position of the theophylline molecule produced molecular structures which were generally in favor of adenosine receptor antagonism. This is evident from the potency order of 8-substituted theophyllines as adenosine receptor antagonists: 8-(p-bromophenyl)theophylline, 8-(p-methylphenyl)theophylline, 8-phenyltheophylline and 8-(p-chlorophenyl)theophylline, 8-(methoxy-phenyl)theophylline > 8-(dimethylaminophenyl)-theophylline ? 8-benzyltheophylline > theophylline. The order of potency for inhibition of brain membrane phosphodiesterase was: 1,3-dimethyl-2,6-dithioxopurine > methylxanthines > 8-substituted theophyllines. The results suggest that 8-substituted theophyllines may be more selective in their activity as adenosine receptor antagonists whereas an increase in lipid solubility by substituting at the 1,2,3 and 6 positions of the purine ring may result in an increase in phosphodiesterase inhibition.