亲和层析法分离纯化猪肺血管紧张素转换酶

亲和胶合成实验以双环氧化合物1,4-丁二醇-2-缩水甘油醚(1,4-butanediol diglycidyl ether)为活化体及连接臂,在硼氢化钠(NaBH)存在的碱性条件下,将载体Sepharose CL―4B与雷诺普利(lisinopril)共价连接在一起,成功合成亲和层析胶,并利用亲和层析胶对猪肺血管紧张素转换酶（angiotensin converting enzyme, ACE）进行分离提纯.猪肺组织匀浆经1.6～2.6 mol/L硫酸铵分级沉淀、透析平衡、亲和柱分离等步骤,从200 g猪肺中提纯得到0.79 mg ACE蛋白,酶活力回收11.9％,比活力38.8 U/mg.与层析前的酶液比较,亲和层析一步提纯可达264倍;与肺匀浆液比较提纯达808倍.SDS-聚丙烯酰胺凝胶电泳可见,提纯的猪肺ACE为一条带,分子质量约为180 ku.     

猪肺血管紧张素转换酶的提纯

本文报道了猪肺血管紧张素转换酶(ACE)的提纯方法及其鉴定,并讨论了方法的改进。肺匀浆经1.6—2.6mol/L硫酸铵沉淀,Sephadex G-200凝胶过滤,DEAE-Sephaeel及羟基磷灰石柱层析步骤,从168克肺中获得4.5毫克酶蛋白纯品。活力回收45.2％,比活力15.6单位/毫克蛋白;和匀浆上清比较,提纯390倍。经聚丙烯酰胺凝胶电泳(pH8.3)鉴定为一条带。按SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)测得其分子量为132,000道尔顿。酶蛋白在-30℃貯存10月,比活力丢失30％。     

血管紧张素转换酶2对心脏作用的研究进展

血管紧张素转换酶2作为肾素—血管紧张素系统的新成员,对心脏功能及心脏节律发挥着重要的调节作用。缺乏ACE2会造成心功能的下降,原因可能是心肌慢性缺氧、血管紧张素Ⅱ水平的提高、血管紧张素(1-7)对心脏保护作用的缺失以及其他肽类底物的增加。但同时ACE2的过度表达又会引起心脏传导紊乱和致死性的心律失常。因此,ACE2精确的生理作用有待进一步明确,但调节ACE2的活性可能为心血管疾病的治疗提出了新的思路。本文主要介绍了ACE2的分布与特性,及其对心功能及心脏节律的影响。     

血管紧张素转换酶2-血管紧张素1-7-Mas轴对血管作用的研究进展

血管紧张素转换酶2（ACE2）和Mas受体的发现使人们对肾素-血管紧张素（RAS）有了更全面的认识。ACE2可水解血管紧张素Ⅰ和血管紧张素Ⅱ直接或间接生成血管紧张素1-7（Ang 1-7）,并与高血压的形成密切相关。Ang 1-7主要通过Mas受体引起血管舒张、抑制细胞增殖。ACE2-Ang1-7-Mas轴的发现为RAS的研究、高血压等心血管疾病的防治和新药开发提供了新的思路和方向。     

血管紧张素转换酶的克隆及序列分析

根据GenBank上公布的小鼠血管紧张素转换酶(ACE)基因序列,经多重比较设计1对特异性引物,从小鼠Balb/c肺部组织中提取总RNA,进行RT-PCR。产物经纯化回收,克隆到pMD-18T载体,转化大肠杆菌DH5α感受态细胞,检测阳性克隆、测序并进行序列分析。测序结果显示,该片段全长4 023 bp,开放阅读框(ORF)包含3 985 bp,编码1 328个氨基酸,相对分子质量为152.77 kD,等电点6.35。此序列与GenBank已登录的小鼠ACE同源性达99%,差异的5个碱基位于基因的非关键部位,说明小鼠ACE成功克隆。经同源性比较分析,达50%以上相同性的物种有17个,且符合种属之间的进化关系。因此,该序列可于研究物种亲缘关系或遗传距离的理想标记,为下一步研究其在酵母中的表达、生物活性和应用奠定了一定的基础。     

血管紧张素转换酶2的功能和应用

人血管紧张素转换酶2（ACE2）是肾素-血管紧张素系统（RAS）的重要调节分子,它在控制心血管和血压的正常生理活动中具有重要的作用。此外,ACE2作为SARS病毒的受体,对于病毒的入侵起关键作用。目前ACE2已经被用于高血压和心血管相关疾病的药物靶标设计和基因治疗,随着研究的深入,ACE2在临床上的应用将更加广泛。     

血管紧张素转换酶2在心血管疾病中的调控作用

血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)是肾素-血管紧张素系统(renin-angio-tensin system,RAS)的重要成员,广泛分布于肺、心血管系统、肠道、肾脏、中枢神经系统和脂肪组织中.ACE2具有多种生理功能,在防止高血压、动脉粥样硬化、心力衰竭、肺...     

汉族人群中血管紧张素转换酶抑制剂所致咳嗽与血管紧张素转换酶基因及缓激肽β2受体基因多态性的关系

目的:探讨汉族人群中血管紧张素转换酶抑制剂(ACEI)所致咳嗽与血管紧张素转换酶(ACE)基因及缓激肽β2受体(BDK-RB2)基因多态性的关系。方法:应用聚合酶链反应(PCR)方法,检测汉族人群中151例由于服用ACEI引起的咳嗽患者及151例未发生咳嗽的患者的ACE I/D及BDKRB2 C/T的多态性,并采用紫外法检测ACE活性。结果:发现ACE基因分布在咳嗽组中II型为47.0%,ID型为42.4%,DD型为10.6%;无咳嗽组分别为39.7%、47.0%、13.3%,两组相比其差异具有统计学意义(P<0.01);BDKRB2基因分布在咳嗽组中CC型为21.3%,CT型为50.0%,TT型为28.7%,无咳嗽组分别为22.5%、47.7%、29.8%,两组相比其差异无统计学意义(P>0.05);咳嗽组ACE活性水平为([28.3±10.1)U/L]明显低于无咳嗽组([40.2±9.4)U/L],两组相比其差异具有统计学意义(P<0.01)。结论:汉族人群中ACEI所致咳嗽与ACE基因多态性及血清ACE水平有关,BDKRB2 C/T与咳嗽间未发现有统计学意义的关联。     

从米糠中提取降血管紧张素转换酶抑制剂

采用盐析法提取米糠蛋白,分别选用胃蛋白酶、胰蛋白酶、木瓜蛋白酶等单独或联合水解米糠蛋白,以从酶解米糠蛋白中分离获得具有血管紧张素转换酶（ACE）抑制活性的短肽组分。经Sephadex G-15凝胶层析和SP—Sephadex C-25离子交换层析分离各酶解组分,并检测各组分的ACE抑制活性。结果表明,米糠蛋白的酶解分离产物中含有较强ACE抑制活性的组分,其中经胃蛋白酶和胰蛋白酶共同水解时得到的小分子量寡肽组分的抑制活性最强,为后续对其进行结构分析奠定了基础。     

数种底物同时存在时的血管紧张素转换酶的酶促反应

数种底物同时存在时的血管紧张素转换酶的酶促反应任维栋（潍坊医学院,山东潍坊２６１０４２）关键词血管紧张素转换酶,底物,动力学方程血管紧张素转换酶（Ａｎｇｉｏｔｅｎｓｉｎ－ｃｏｎｖｅｒｔｉｎｇｅｎｚｙｍｅ,简称ＡＣＥ）（ＥＣ３,４,１５．１）,又称肽酰...     

Purification and Characterization of a Peptidyl Dipeptidase Resembling Angiotensin Converting Enzyme from the Electric Organ of Torpedo marmorata

The electric organ of Torpedo marmorata contains a membrane-bound, captopril-sensitive metallopeptidase that resembles mammalian angiotensin converting enzyme (peptidyl dipeptidase A; EC 3.4.15.1). The Torpedo enzyme has now been purified to apparent homogeneity from electric organ by a procedure involving affinity chromatography using the selective inhibitor lisinopril immobilised to Sepharose via a 28-A spacer arm. The purified protein, like the mammalian enzyme, acted as a peptidyl dipeptidase in cleaving dipeptides from the C-terminus of a variety of peptide substrates, including angiotensin I, bradykinin, [Met5]enkephalin, [Leu5]enkephalin, and the model substrate hippuryl (benzoylglycyl; BzGly)-His-Leu. The hydrolysis of BzGly-His-Leu was activated by Cl-. Enzyme activity was inhibited by classical angiotensin converting enzyme inhibitors, including captopril, enalaprilat (MK422), and lisinopril (MK521). Torpedo angiotensin converting enzyme, like its mammalian counterpart, was also able to act as an endopeptidase in hydrolysing the amidated neuropeptide substance P. Hydrolysis of substance P occurred primarily at the Phe8-Gly9 bond with release of the C-terminal tripeptide, Gly-Leu-MetNH2, and this hydrolysis was blocked by selective inhibitors. The Torpedo enzyme was recognised by a polyclonal antibody to pig kidney angiotensin converting enzyme on immunoelectrophoretic (Western) blot analysis. Thus, on the basis of substrate specificity, inhibitor sensitivity, and immunological criteria, the Torpedo enzyme closely resembles mammalian angiotensin converting enzyme. However, the Torpedo enzyme appears somewhat larger (Mr = 190,000) than the pig kidney enzyme (Mr = 180,000) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The endogenous peptide substrate(s) for Torpedo electric organ angiotensin converting enzyme and the physiological role of the enzyme in this tissue remain to be evaluated.     

Characterization of Neuronal and Endothelial Forms of Angiotensin Converting Enzyme in Pig Brain

The molecular forms of angiotensin converting enzyme (ACE; EC 3.4.15.1) in preparations of pig brain cortical microvessels and striatal synaptosomal membranes have been identified by immunoelectrophoretic blot analysis. The cortical microvessels contained only the endothelial form of the enzyme, Mr 180,000, which comigrated with pig kidney ACE on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, the synaptosomal membranes contained only a smaller form of ACE, Mr 170,000, which represents the neuronal form of the enzyme. No significant differences in inhibitor sensitivity or substrate specificity were detected between the two forms of ACE. In particular, neurokinin A was resistant to hydrolysis by either microvessel or synaptosomal membrane ACE, and the pattern of hydrolysis of substance P by the two preparations was identical.     

Characterization of New Milk-derived Inhibitors of Angiotensin Converting Enzyme In Vitro and In Vivo

Inhibition of angiotensin converting enzyme (ACE) has been observed with a variety of different peptides, and peptide fragments with inhibitory capabilities have been identified within many different proteins, including milk proteins. The purpose of this study therefore was to identify new short peptides with inhibitory properties from the primary structure of milk proteins and to characterize them in vitro and in vivo, since no milk derived ACE inhibitors have previously been evaluated for their ability to inhibit ACE in vivo. In vitro, 8 of 9 dipeptides were found to be competitive inhibitors of ACE. The IC₅₀ was significantly lower when an angiotensin I-like substrate was used, than when a bradykinin-like substrate was used. Using three different in vivo models for ACE inhibition, a very moderate effect was observed for three of the new peptides, but only for up to 6 or 12 minutes. Nothing was observed with two reference compounds that are reported to be hypotensive ACE-inhibitors derived from milk proteins. This raises the question whether the mechanism of hypotensive action is straightforward inhibition of ACE in vivo.     

Angiotensin Converting Enzyme in Alzheimer''s Disease: Increased Activity in Caudate Nucleus and Cortical Areas

The activity of the dipeptidyl carboxypeptidase, angiotensin converting enzyme, was assayed in several brain regions of patients dying with Alzheimer's disease and compared to that of appropriately age-matched controls. Enzyme activity was found to be elevated by 44% and 41% in the medial hippocampus and parahippocampal gyrus, respectively, and by 27% and 29% in the frontal cortex (area 10 of Brodman) and caudate nucleus, respectively, in Alzheimer's disease patients. Converting enzyme activity did not differ from controls in the nucleus accumbens, substantia nigra, temporal cortex, anterior or posterior hippocampus, amydgala, and septal nuclei.     

血管紧张素转换酶的结构功能及相关抑制剂

血管紧张素转化酶(angiotensin converting enzyme, ACE, EC 3.4.15.1)是一种位于细胞膜上, 依赖锌离子的羧二肽酶, 催化水解十肽血管紧张素I羧基末端两个氨基酸, 生成具有血管收缩作用的八肽血管紧张素II。ACE在血压调节系统renin - angiotensin system (RAS系统)中具有重要作用, 从ACE的结构功能、基因多态性及其抑制剂等方面进行了详细综述。发现体细胞ACE两个活性中心催化血管紧张素I和缓激肽的机制不同, 因此以体细胞ACE单个活性中心为靶点的研究, 将会为研制开发副作用更少, 安全性更高的ACE抑制剂提供新的途径。     

Ketomethylureas. A New Class of Angiotensin Converting Enzyme Inhibitors

Abstract

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term “ketomethylureas”, is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE wtih I₅₀ values in the nM range.     

Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Angiotensin Converting Enzyme-2 (ACE2) in Human Urine

Angiotensin converting enzyme-2 (ACE2) is a recently described membrane-bound carboxypeptidase identified by its homology to ACE, the enzyme responsible for the formation of the potent vasoconstrictor angiotensin II (Ang II). ACE2 inactivates Ang II and is thus thought to act in a counter-regulatory fashion to ACE. ACE2 is highly expressed in epithelial cells of distal renal tubules, and recent evidence indicates that expression is increased in a range of renal diseases. A soluble form of ACE, generated by proteolytic cleavage of the membrane-bound form, has been shown to be present in urine; although evidence for a similar release of ACE2 has been reported in cell culture, it is not yet known whether this occurs in vivo. The present study has identified ACE2 in human urine, both by a sensitive fluorescence-based activity assay and by Western immunoblot. Levels of ACE2 were surprisingly higher than ACE, which may reflect preferential targeting of the enzyme to the luminal surface of the renal epithelium. Future studies will determine whether increased expression of ACE2 in renal diseases are reflected in higher urinary levels of this novel enzyme.Australian Peptide Conference Issue.     

Oxidant Stress and Glomerular Prostanoid Production: Influence of Angiotensin Converting Enzyme Inhibition

1. The effect of H₂O₂ (4.7 × 10^-9 4.7 × 10^-3M) on prostanoid production by isolated glomeruli from normotensive (WKY) and, spontaneously hypertensive rats (SHR) has been studied.

2. Oxidant stress significantly increased synthesis of prostaglandin E₂(PGE₂), I₂(PGI₂)and thromboxane A₂ (TxA₂) by glomeruli from both strains whereas the ratio (PGE₂ + PGI₂)/TxA₂ increased in only SHR.

3. Pre-incubation of glomeruli with the angiotensin converting enzyme inhibitors captopril or lisinopril, had virtually no effect on H₂O₂-induced synthesis of individual prostanoids nor on the ratio (PGE₂ + PGI₂)/TA₂ by glomeruli from either WKY or SHR.

4. The findings suggest that H₂O₂-induced changes in glomerular function may be mediated, in part, by PGs but fail to support the suggestion that the ability of ACEI to protect glomeruli from H₂O₂-induced damage is determined by PGs.     

Angiotensin Converting Enzyme Inhibitors as Oxygen Free Radical Scavengers

The authors have compared the ability of two non-SH-containing angiotensin converting enzyme (ACE) inhibitors (enalaprilat and lisinopril) with an -SH containing ACE inhibitor (captopril) to scavenge the hydroxyl radical (OH). All three compounds were able to scavenge -OH radicals generated in free solution at approximately diffusion-controled rates (10¹⁰ M^-1s^-1) as established by the deoxyribose assay in the presence of EDTA. The compounds also inhibited deoxyribose degradation in reaction mixtures which did not contain EDTA but not so effectively. This later finding also suggests that they have some degree of metal-binding capability. Chemiluminescence assays of oxidation of hypoxanthine by xanthine oxidase in the presence of luminol, confirm that the three ACE inhibitors are oxygen free radical scavengers. Our results indicate that the presence of a sulphydryl group in the chemical structure of ACE inhibitors is not relevant for their oxygen free radical scavenging ability.