降钙素基因相关肽——一种强烈的舒血管物质

由37个氨基酸组成的新肽,降钙素基因相关肽(CGRP)是降钙素基因原始RNA 转录的产物之一。通过降钙素基因密码序列的分析,得到了大鼠CGRP 的一级结构,并已人工合成。人CGRP 亦被分离和鉴定,其组成亦为37个氨基酸,有四个和大鼠CGRP 不同。CGRP C 末端免疫活性已在大鼠中枢和外周神经系统中测得,大鼠主动脉提取物也含有免疫活性CGRP(20pmol/g)。离体大鼠三叉神经节能释放免疫活性CGRP,高K~ 浓度使之释放增加。有证据表明,CGRP 是中枢和外周神经系统递质,对心脏有强烈作用,脑     

肾上腺髓质素前体中间片段——急性心肌梗死患者预后标志分子

肾上腺髓质素（adrenomedullin,ADM）是1993年由日本学者Kitamura等从人的嗜铬细胞瘤组织中发现并分离的一种含52个氨基酸残基的心血管活性多肽,属于降钙素基因相关肽超家族。     

人骨形成蛋白-2C端102肽的诱骨活性

为分析更短的Hbmp-2C端肽是否具有诱骨活性 ,寻求新型的有诱骨活性的基因工程Hbmp-2产品。利用温度诱导的大肠杆菌表达系统表达肽段长度为102个氨基酸的Hbmp-2C端肽及其Cys的突变体。表达产物经纯化复性后 ,植入小鼠肌袋模型中测试其诱骨活性。获得了能稳定表达Hbmp 2C端肽的工程菌 ,测序结果与预期的序列完全一致。表达产物以包涵体形式存在 ,表达量占细胞总蛋白的 3 0 %。产物经纯化复性后 ,小鼠肌袋模型测试结果表明 :Hbmp 2 10 2肽仍具有诱骨活性 ,而将C端第一位Cys突变的 10 2肽诱骨活性丧失。实验表明 :比Hbmp 2成熟肽 ( 114个氨基酸 )更短的C端 10 2肽仍具有良好诱骨活性 ,这 10 2肽N端第一个Cys对其诱骨活性可能是必需的。     

人肽抗生素hPAB—β分子的同源模建及其突变体设计

从人体基因组中克隆了一个编码肽抗生素样的基因。按基因编码产物的氨基酸序列 ,化学合成目的产物 ,经药物敏感测定法证实产物具有杀菌活性。旨在通过同源模建的方法 ,构建肽抗生素hPAB β的突变体分子 ,希望获得肽链更短 ,但生物活性不降低甚至更强的突变体。方法是在序列比较的基础上 ,利用同源分子对目的肽抗生素hPAB β进行同源模建 ,在此模型上 ,删除某些氨基酸后观察目的分子立体结构模型改变情况 ,从而确定突变体分子结构。同源模建结果表明hPAB β由一个α螺旋和 3个 β片层构成 ,二级结构保守 ;具有两亲性结构 ,推测与目的肽进…     

人α降钙素基因相关肽基因在酵母细胞中的分泌表达与产物的分离纯化

化学合成的人α降钙素基因相关肽(CCRP)基因用PCR法改造后使其能正确融合在酵母分泌型表达载体pVT102U/α中的α交配因子前导肽序列之后,然后进行克隆并转化酵母宿主菌S－78进行表达．培养物的上清用酶标(ELlSA)鉴定为阳性,而对照S－78、pVT102u／α为阴性,表达量用ELISA定量大于2mg／L。表达产物经阳离子交按层析(CM—Sphadex C₂₅)和HPLC纯化得到了HPLC纯产品。纯化后的CGRP能引起小鼠血压的降低,说明表达的目的蛋白既有CGRP的免疫结合活性,又有CGRP的生理活性。测定其N－端10个氨基酸序列,证明人工合成的CGRP基因在酵母细胞中已正确表达。     

人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶联表达

人降钙素 (hCT)是 32氨基酸的多肽激素 ,C-端为α脯氨酰胺结构 ,具有调节体内钙、磷代谢等许多重要生理功能。用重组昆虫杆状病毒表达系统 ,偶联表达合成的人修饰型降钙素 (hmCT)基因与GST融合基因和大鼠酰胺化酶 (PAM)基因 ,再用抗hmCT或抗PAM抗体 ,既检测到由昆虫细胞表达的GSThmCT产物也检测到PAM产物。经GSH 琼脂糖凝胶亲和层析 ,分离纯化GSThmCT融合蛋白。这种蛋白修饰酶与底物在真核细胞偶联表达也将适用于其他生物活性肽的体外表达     

内毒素结合肽的原核表达、纯化及生物学活性鉴定

重组人内毒素结合肽 (endotoxinbindingpeptide ,EBP)融合蛋白在大肠杆菌中表达 ,分离和纯化后对其进行生物学活性观察 .将构建好的PinpointⅩa3 EBP生物素融合表达载体转化大肠杆菌DH5α ,IPTG诱导表达菌株 ,亲和层析法纯化表达产物 ,因子Ⅹa(factorⅩa)切割分离内毒素结合肽 ,采用凝胶过滤和反相液相高效色谱法两步纯化 ,从相对分子质量、N端 1 0个氨基酸的序列分析等方面进行鉴定 ;利用人单核细胞U937对重组内毒素结合肽进行了生物学活性的检测 .结果发现 ,内毒素结合肽以包涵体形式存在 ,因子Ⅹa酶切融合蛋白后得到 3 5kD的内毒素结合肽 ,纯化后内毒素结合肽纯度达 99%以上 ,N端 1 0个氨基酸的分析结果与预期相符 ;初步证实内毒素结合肽具有较好的LPS结合活性 ,能够抑制LPS的作用 .经原核表达及纯化复性 ,获得了具有较好生物学活性的内毒素结合肽 ,为进一步研究其功能奠定了良好的基础     

降钙素基因相关肽家族的受体活性修饰蛋白

降钙素基因相关肽家族中的降钙素、胰淀粉样酶、两种降钙素基因相关肽和肾上腺髓质素具有相似的结构。受体活性的修饰蛋白（RAMP）是新近从蟾蜍卵细胞中发现并克隆出来的蛋白质。受体活性修饰蛋白是具有单一跨膜功能域的蛋白,可调节降钙素的受体样受体（CRLR）向细胞膜的转运和识别配体的特异性。不同的RAMP可与降钙素受体试样受体或降钙素受体结合表现为对不同配体具有亲和的、不同的受体表型而决定了体内的生物学效应。RAMP1转运的末端糖基化的成熟的CRLR蛋白,使CRLR表现为功能性的降钙素基因相关肽（CGRP）受体表型;RAMP2转运的CRLP是核心糖基化的未成熟的CRLR蛋白,使CRLR表现为功能性的肾上腺髓质素（Adm）受体表型。RAMP亦可与降钙素受体作用产生Amylin受体表型。     

蚯蚓纤溶酶基因P239的原核表达、纯化及活性测定

通过RT-PCR方法从蚯蚓(Lumbricus bimastus)中获得蚯蚓纤溶酶基因,命名为P-239,含有 852 个核苷酸,成熟肽编码 239 个氨基酸,通过Genbank 序列同源性比较,与已知的蚯蚓纤溶酶有一定的同源性,为首次获得的新基因.随后构建了pBV-220/P-239重组质粒,在大肠杆菌DH5α中进行原核表达,再经亲和层析柱纯化复性,其产物经活性测定,确定不仅具有激酶作用,而且具有直接溶栓活性.     

棕点湍蛙皮肤分泌液中促胰岛素释放肽的分离纯化、基因克隆及活性分析

通过分离纯化棕点湍蛙(Amolops loloensis)皮肤分泌液中的生物活性物质,得到有促胰岛素释放活性的分离峰,并鉴定其结构.采用葡聚糖Sephadex G-50凝胶层析和反相高效液相(RP-HPLC)等手段对棕点湍蛙皮肤分泌液进行分离纯化,利用胰岛素释放实验进行活性检测,Edman降解法测定活性峰的氨基酸序列,反转录法构建cDNA文库并克隆其基因.得到一个具有显著的促胰岛素释放活性的十六肽,测得其氨基酸序列为:FMPIvGKsMSGLSGKL-NH2,命名为amolopin-1.由cDNA(开放阅读框为192bp)推导的氨基酸一级结构显示,其前体由64个氨基酸残基(aa)组成,包括高度保守的信号肽(22aa),酸性肽以及成熟肽.经过数据库序列比对,从棕点湍蛙皮肤中得到一个新的促胰岛素释放肽,进一步分析其作用机理和药代动力学,极有可能得到一个新的治疗糖尿病的降糖药物.     

The importance of the amino acid in position 32 of cholecystokinin in determining its interaction with cholecystokinin receptors on pancreatic acini

In the C-terminal heptapeptide of cholecystokinin, replacement of the penultimate residue, aspartic acid, by beta-alanine caused a 300-fold decrease in the potency with which the peptide stimulated enzyme secretions, whereas replacement by glutamic acid caused a 1000-fold decrease in potency. The beta-alanine-substituted peptide was approximately ten times more potent when the n terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl, and the glutamic acid-substituted peptide was approximately twice as potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl. Changes in the ability of the peptide to stimulate amylase secretion were accompanied by corresponding changes in the ability of the peptide to inhibit binding of 125I-labeled cholecystokinin. The magnitude of stimulation of enzyme secretion caused by a maximally effective peptide concentration was the same with each analogue as it was with the unaltered peptide. Replacing the aspartyl residue by beta-alanine or glutamic acid or replacing the N-terminal t-butyloxycarbonyl moiety by benzyloxycarbonyl caused an equivalent decrease in the ability of the peptide to stimulate enzyme secretion and its ability to cause residual stimulation of enzyme secretion. In contrast, the N-terminal desamino analogue of cholecystokinin heptapeptide was ten times less potent than the unaltered peptide in stimulating amylase secretion, but 100 times less potent than the unaltered peptide in causing residual stimulation of enzyme secretion.     

Isolation and sequence determination of an active site peptide of rabbit muscle pyruvate kinase

Rabbit muscle pyruvate kinase was inactivated by 2', 3'-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide is identical to a tryptic peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.     

The importance of the amino acid in position 32 of cholecystokinin in determining its interaction with cholecystokinin receptors on pancreatic acini

In the C-terminal heptapeptide of cholecystokinin, replacement of the penultimate residue, aspartic acid, by β-alanine caused a 300-fold decrease in potency with which the peptide stimulated enzyme secretion, whereas replacement by glutamic acid caused a 1000-fold decrease in potency. The β-alanine-substituted peptide was approximately ten times more potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl, and the glutamic acid-substituted peptide was approximately twice as potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl. Changes in the ability of the peptide to stimulate amylase secretion were acompanied by corresponding changes in the ability of the peptide to inhibit binding of ¹²⁵I-labeled cholecystokinin. The magnitude of stimulation of enzyme secretion caused by a maximally effective peptide concentration was the same with each analogue as it was with the unaltered peptide. Rpelacing the aspartyl by β-alanine or glutamic acid or replacing of N-terminal t-butyloxycarbonyl moiety by benzyloxycarbonyl caused an equivalent decrease in the ability of the peptide to stimulate enzyme secretion and its ability to cause residual stimulation of enzyme secretion. In contrast, the N-terminal desamino analogue of cholecystokinin heptapeptide was ten times less potent than the unaltered peptide in stimulating amylase secretion, but 100 times less potent that the unaltered peptide in causing residual stimulation of enzyme secretion.     

A polyamine-conjugated peptide isolated from human plasma

When human plasma was fractionated by gel exclusion chromatography on Bio-Gel P-10, substantial quantities of putrescine and trace amounts of spermidine were consistently associated with a 4200 M_r peptide species. Putrescine was not removed from the peptide by extensive dialysis, desalting, multiple gel and ion exchange chromatographic procedures, nor by incubation with urea followed by trichloroacetic acid precipitation. No putrescine was detected unless the peptide was acid hydrolyzed. Incubation of plasma with ¹⁴C-labeled putrescine did not result in association of the label with the peptide. We conclude that these polyamines appear to be covalently bound to this major plasma peptide species. The amount of putrescine associated with the peptide is 10- to 40-fold the concentration of unbound plasma putrescine per equivalent amount of plasma. The peptide was purified to apparent homogeneity and was found to be a single chain composed of 32 amino acid residues with a combined molecular weight of 4180. Possible biological roles of this polyamine conjugated peptide are discussed.     

Semisynthetic analogs of cytochrome c reconstructed from natural and synthetic peptides.

A biologically active semisynthetic hybrid of horse heart cytochrome c has been prepared by combining the heme peptide 1 through 65 (HP 1-65), prepared by CNBr cleavage of natural cytochrome c, with a semisynthetic peptide corresponding to positions 66 through 104. A fully protected synthetic peptide 66--79 was prepared by a modified solid phase peptide synthesis procedure and was converted to its N-hydroxysuccinimide ester. A peptide corresponding to residues 81--104 of cytochrome c was also isolated from the CNBr cleavage mixture and its epsilon-amino groups and tyrosyl hydroxyl group were protected selectively with the t-butyloxycarbonyl group. This partially protected peptide was reacted with t-butyloxycarbonyl methionine N-hydroxysuccinimide ester to give a derivative having methionine at position 80. This product was deprotected, purified and then t-butyloxycarbonyl groups were again introduced specifically on the epsilon-amino groups to give the peptide, Boc(Lys,Tyr)80--104. A semisynthetic peptide corresponding to residues 66 through 104 of cytochrome c was prepared by condensing the synthetic peptide 66--79 N-hydroxysuccinimide ester with t-butyloxycarbonyl (Lys,Tyr)80--104. The semisynthetic product was deprotected, purified and combined under anaerobic conditions with a heme peptide, HP 1-65, that was isolated from the products of CNBr cleavage of native cytochrome c. The reconstituted semisynthetic cytochrome c was purified by ion exchange chromatography and was shown to have the same oxygen uptake as native cytochrome c when assayed in the succinate oxidase system.     

Cyanogen bromide digestion of the avian myeloblastosis virus pp19 protein: isolation of an amino-terminal peptide that binds to viral RNA.

The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.     

Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase. Characterization of the peptide and its role in the interaction of reductase with cytochrome P-450

A peptide identified as the membrane-associated segment of NADPH-cytochrome P-450 reductase has been generated by steapsin protease treatment of vesicle-incorporated reductase and isolated by preparative gel electrophoresis. This peptide remains associated with vesicles when steapsin protease digests of vesicle-incorporated reductase were fractionated by Sepharose 4B chromatography, confirming its identity as the membrane-binding peptide. The molecular weight of the membrane-binding peptide was 6400 as determined by gel filtration in 8 M guanidine hydrochloride, and its amino acid content was not especially hydrophobic. The activity of reconstituted hydroxylation systems consisting of reductase, cytochrome P-446, and dilauroyl phosphatidylcholine was not inhibited by large molar excesses of purified membrane-binding peptide. Moreover, when purified reductase and cytochrome P-446 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptides were used. Similar results were obtained with reductase, cytochrome P-450, and detergent-solubilized liposomes (with or without the membrane-binding peptide). Thus, the membrane-binding peptide does not appear to interact with either of these two forms of the hemoprotein in a site-specific manner to prevent reconstitution of hydroxylation activity.     

Changes in lipid mobility associated with alamethicin incorporation into membranes

The binding state of the antibiotic peptide alamethicin with phospholipid bilayers was investigated in terms of the changes induced in lipid mobility. Fluorescence anisotropy was used for the study. It was found that an increase in peptide concentration induced different changes in lipid mobility above and below a critical peptide concentration. This concentration was also critical for an increase in the cooperative binding of the peptide, as detected by circular dichroism. Above the critical peptide concentration, the mobility of both lipid regions, around the polar head and hydrocarbon chain, became restricted with an increased peptide concentration. Below the critical level, however, an increased peptide concentration induced a "wobbling" of the lipid hydrocarbon chain. These results show that an increase in the cooperative binding of the peptide is accompanied by a change in the dominant configuration of the binding peptide. When the binding peptide increases, the dominant configuration appears to shift from surface association to deep incorporation within the membrane. This shift in configuration means that in the formation of ion-conductive pores, voltage-driven insertion of the peptide is a prominent step below a critical peptide concentration.     

Incorporation of a synthetic mitochondrial signal peptide into charged and uncharged phospholipid monolayers

The interaction of the chemically synthesized 25-residue signal peptide of subunit IV of yeast cytochrome c oxidase with synthetic and natural phospholipids was studied by using a monolayer technique. Incorporation of the peptide into phospholipid monolayers was measured as surface area increase at constant surface pressure. The peptide was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into phospholipid monolayers up to limiting pressures of 30-40 mN/m. The incorporation of the positively charged peptide was strongly enhanced by the presence of negatively charged phospholipids. The molecular area of the signal peptide in monolayers was determined with a 14C-labeled signal peptide and was 560 +/- 170 A2. This is consistent with a 25-residue alpha-helical peptide incorporating with its long axis parallel to the plane of the monolayer. Incorporation isotherms into synthetic phosphatidylcholine and phosphatidylglycerol monolayers at different charge densities were analyzed in terms of a simple incorporation/binding model, involving partitioning of the peptide into the monolayer and an in-plane binding reaction of the negatively charged phospholipids to the partitioned peptide.     

Effect of insulin on ATP-citrate lyase phosphorylation: regulation of peptide A and peptide B phosphorylations

Insulin decreases multifunctional protein kinase (MFPK) activity in rat adipose tissue [Ramakrishna, S., & Benjamin, W. B. (1988) J. Biol. Chem. 263, 12677-12681]. Insulin also decreases the phosphorylation of peptide B but increases the phosphorylation of peptide A of ATP-citrate lyase (ATP-CL). The mechanism for this increase in peptide A phosphorylation was studied with purified ATP-CL from control and insulin- and isoproterenol-treated fat pads by using MFPK and the catalytic subunit of cAMP-dependent protein kinase (A-kinase). ATP-CL purified from insulin-treated fat pads is a better substrate for phosphorylation by MFPK compared to controls. This result is consistent with the hypothesis that insulin action decreases peptide B phosphorylation. To determine if the degree of phosphorylation at peptide B affects the phosphorylation rate of peptide A by A-kinase, ATP-CL was prepared with determined phosphate contents of peptides A and B. ATP-CL with a low phosphate content at peptide B is a better substrate for phosphorylation at peptide A by A-kinase than is ATP-CL with a high phosphate content at peptide B. These results suggest that the insulin-induced increase in ATP-CL phosphorylation at peptide A is due to a decrease in peptide B phosphorylation. ATP-CL prepared from isoproterenol-treated fat pads is also a better substrate for phosphorylation at peptide B by MFPK than controls. This increase in phosphorylation at peptide B by MFPK is due to positive second-site regulation by the isoproterenol-induced increase in peptide A phosphorylation.