微生物来源的HLE抑制剂N01WA-735E的研究

人白细胞弹性蛋白酶抑制剂为筛选炎症和癌症的重要靶点。应用白细胞弹性蛋白酶抑制剂高通量的筛选模型对数千株放线菌进行筛选,发现了阳性菌株N01WA-735。首先通过形态学和化学分类学鉴定其为链霉菌属。采用有机溶剂提取、硅胶柱色谱、Sephadex LH-20柱色谱和结晶等方法对该菌株的发酵产物进行了分离纯化,得到活性单体化合物N01WA-735E,通过对N01WA-735E的理化性质和波谱数据分析,确定其结构与文献报道的化合物BE-52440A相同。该化合物对人白细胞弹性蛋白酶有很强的抑制活性,其IC50为3.02μmol/L。该化合物对人白细胞弹性蛋白酶的抑制活性国内外未见报道。     

微生物来源的HLE抑制剂N01WA-735E的研究

人白细胞弹性蛋白酶抑制剂为筛选炎症和癌症的重要靶点。应用白细胞弹性蛋白酶抑制剂高通量的筛选模型对数千株放线菌进行筛选,发现了阳性菌株N01 WA-735。首先通过形态学和化学分类学鉴定其为链霉菌属。采用有机溶剂提取、硅胶柱色谱、Sephadex LH-20柱色谱和结晶等方法对该菌株的发酵产物进行了分离纯化,得到活性单体化合物N01 WA-735E,通过对N01 WA-735E的理化性质和波谱数据分析,确定其结构与文献报道的化合物BE-52440A相同。该化合物对人白细胞弹性蛋白酶有很强的抑制活性,其IC50为3.02μmol/L。该化合物对人白细胞弹性蛋白酶的抑制活性国内外未见报道。     

蛋白酶抑制剂1O—Ov-抑丝酶家族的新成员

抑丝酶(serine proteinase inhibitor,ser-pin)是一类与α1-蛋白酶抑制剂(α1-PI)结构相类似的蛋白质组成的超家族,在一系列重要的生理病理过程中,如凝血、纤溶、补体活化、感染、细胞迁移等,发挥着重要作用。其中,鸡卵清蛋白亚家族又代表了一系列结构更为相似的抑丝酶蛋白,纤溶酶原激活剂抑制剂2(PAI-2)、人白细胞弹性蛋白酶抑制剂(human leukocyte elastase inhibitor,HLEI)、胞浆抗蛋白酶(CAP)、蛋白酶抑制剂6(PI-6)、蛋白酶抑制剂9(PI-9)等都是该家族的典型成员。1995年人们又从人骨髓细胞eDNA文库中克隆出了一个新基因Bomapin(bonemarrow associated serpin),该基因编码一个分子量为45 kD的蛋白质,被基因组数据库委员会命名为蛋白酶抑制剂10(PI-1O)[1],同源性分析表明它是Ov-抑丝酶家族的新成员。     

蛋白酶抑制剂10——Ov-抑丝酶家族的新成员

抑丝酶 (ｓｅｒｉｎｅｐｒｏｔｅｉｎａｓｅｉｎｈｉｂｉｔｏｒ,ｓｅｒ ｐｉｎ)是一类与α1 蛋白酶抑制剂 (α1 ＰＩ)结构相类似的蛋白质组成的超家族 ,在一系列重要的生理病理过程中 ,如凝血、纤溶、补体活化、感染、细胞迁移等 ,发挥着重要作用。其中 ,鸡卵清蛋白亚家族又代表了一系列结构更为相似的抑丝酶蛋白 ,纤溶酶原激活剂抑制剂 2 (ＰＡＩ 2 )、人白细胞弹性蛋白酶抑制剂(ｈｕｍａｎｌｅｕｋｏｃｙｔｅｅｌａｓｔａｓｅｉｎｈｉｂｉｔｏｒ,ＨＬＥＩ)、胞浆抗蛋白酶 (ＣＡＰ)、蛋白酶抑制剂 6 (ＰＩ 6 )、蛋白酶抑制剂 9(ＰＩ 9)等都…     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

旨在克隆、表达与纯化类弹性蛋白多肽,并测定类弹性蛋白的相变温度对不同的盐敏感程度。从头设计了类弹性蛋白多肽的序列并人工合成其编码基因片段,克隆至改造后的表达载体pET-22b中,构建重组表达载体,转化至大肠杆菌BL21(DE3) 中并诱导表达,采用可逆相变循环经高速离心对其进行纯化,并考察了盐类型及浓度对类弹性蛋白相变温度的影响。结果表明：0.4 mmol/L的Na2CO3能使25 μmol/L类弹性蛋白多肽 [KV8F-20] 相变温度降低至19 ℃,此类弹性蛋白多肽序列有望开发成一新型纯化标签,为今后     

绿僵菌分解昆虫外壳蛋白酶MAP-21的纯化与特性

以蝉蜕为底物诱导绿僵菌产生分解昆虫外壳蛋白酶 。发酵液经超滤、Ultrogel AcA 54凝胶层析、制备IEF电泳,纯化了一种蛋白酶MAP-21,SDS-PAGE电泳后经银染色呈单带。该酶的Mr为27kD左右,pI为76。它的特异识别氨基酸为Arg,其活性可被PMSF和TLCK抑制,表明其活性中心有Ser和His残基。它还可被胰蛋白酶的典型抑制剂Leupeptin、Antipain及STI等所抑制,而胰凝乳蛋白酶抑制剂TPCK和胰凝乳弹性蛋白酶抑制剂TEI对其活性无影响。专一底物和抑制剂特性试验结果表明MAP-21是类胰蛋白酶。此外,该酶还可被EDTA所抑制,表明金属离子为其活性所必需。另外还研究了MAP-21的最适作用温度和pH,以及温度耐受性等特性。     

子宫颈机能不全孕妇血清松弛素、弹性蛋白表达意义及对妊娠结局的评估价值研究

摘要 目的：分析子宫颈机能不全孕妇血清松弛素、弹性蛋白表达意义及对妊娠结局的评估价值。方法：选择我院于2020年5月至2022年5月接诊的120例子宫颈机能不全孕妇作为观察组，另选同期的120例正常妊娠孕妇作为对照组。分析血清松弛素、弹性蛋白表达水平，及其与子宫颈机能不全孕妇宫口扩张宽度、宫颈长度的关系，观察血清松弛素、弹性蛋白在早产者与非早产者间的差异性，使用受试者工作特征曲线(ROC)下面积(AUC)评价血清松弛素联合弹性蛋白对早产及围产儿死亡的预测效能。结果：观察组血清松弛素水平较对照组高，弹性蛋白水平较对照组低（P＜0.05）；经Pearson相关性分析，子宫颈机能不全孕妇宫口扩张宽度与血清松弛素水平呈正相关，与弹性蛋白水平呈负相关（P＜0.05）；宫颈长度与血清松弛素水平呈负相关，与弹性蛋白水平呈正相关（P＜0.05）；在120例子宫颈机能不全孕妇中，早产42例、非早产78例；早产组血清松弛素水平高于非早产组，弹性蛋白水平低于非早产组（P＜0.05）；经ROC曲线分析，血清松弛素联合弹性蛋白预测子宫颈机能不全孕妇发生早产的AUC为0.910，预测围产儿死亡的AUC为0.943。结论：血清松弛素水平升高和弹性蛋白水平降低均与子宫颈机能不全有关，两者联合预测早产及围产儿死亡的效能较好，值得进一步研究应用。     

类弹性蛋白多肽及其在生物医学材料的应用

类弹性蛋白多肽是一种人造基因工程蛋白质聚合物,其结构主要由五肽重复串连序列单元 (GVGXP) 的这一肽段单元重复组成。由于具有可逆相变特征,并可进行高通量生产,加之良好的生物相容性及生物可降解性,使其在新型生物医学材料方面展示了广阔的应用前景。概括了类弹性蛋白多肽的相变机理、合成方法及在生物医学材料上的应用,重点阐述了类弹性蛋白多肽在组织工程、靶向肿瘤、构造药物载体微粒的应用。     

球孢白僵菌凝乳弹性蛋白酶(BbPr1)的纯化与特性*

以几丁质为底物,加入基本盐培养基中,诱导球孢白僵菌（Beauveriabassiana）产生分解昆虫表皮的蛋白酶。诱导物中,蝉蜕诱导的蛋白酶总活性、比活较高,经超滤、离子交换层析、亲和层析、制备性IEF电洗脱纯化了一种有凝乳弹性蛋白酶（Pr1）活性的蛋白酶BbPr1。经SDS－PAGE电泳银染后是单带,HPLC凝胶过滤显示单峰。BbPr1为单体酶,分子量为33.6kD左右,pI为7.4。底物专一性测定显示,BbPr1能水解Phe或Leu形成的酸胺键和肽键。BbPrl可被PMSF抑制,表明其活性中心有Ser残基;BbPr1还可被胰凝乳蛋白酶抑制剂TPCK和凝乳弹性蛋白酶抑制剂TEI等所抑制;胰蛋白酶抑制剂beupeptin和Epianstatin,及胃蛋白酶抑制剂Pepstain对BbPr1活性无影响。还研究了BbPr1的最适作用pH和pH稳定性。     

弹性蛋白是一个富饶的有待开发的生物纤维资源

一些生物工程公司都热衷于运用遗传工程菌制造药物,而遗传工程的应用天地是很广的。以弹性蛋白这个生物纤维原料为例,鉴于石油价格不定,这些生物纤维有与石油为原料的市售人造丝和塑料的竞争力。节枝弹性蛋白存在于昆虫羽翼和跳蚤腿部,是一种贮存力能的蛋白质。胶原蛋白和弹性蛋白是构建血管的主要成分。前     

Inhibition by elastase inhibitors of the formyl Met Leu Phe-induced chemotaxis of rat polymorphonuclear leukocytes

Rat leukocyte elastase has been purified successively by AH-Sepharose Kappa-elastin affinity chromatography and by ion exchange chromatography on a carboxymethyl Sephadex resin. It has great similarities with human leukocyte elastase in its molecular weight, substrate specificity and inhibitory profile. The effect of rat leukocyte elastase inhibitors in influencing the chemotactic response of rat PMN to fMetLeuPhe has been compared to that of other proteinase inhibitors. The results indicated that oleoyl (Ala)2ProValCH2Cl, a specific inhibitor of human and rat leukocyte elastases and Eglin C, which also inhibits human and rat cathepsin G, are among the powerful inhibitors of rat PMN chemotaxis induced by the formyl oligopeptide. This suggests that these neutral proteinases, in addition to their known participation in connective tissue catabolism, could play a role in PMN locomotion and chemotaxis.     

Cathepsin-G and leukocyte elastase inactivate human tumor necrosis factor and lymphotoxin

The addition of either cathepsin-G or leukocyte elastase to endotoxin-stimulated human peripheral blood monocytes decreased the immunoreactive tumor necrosis factor (TNF) detected in culture supernatants in a concentration-dependent manner. Both enzymes also induced a loss of supernatant cytolytic activity as determined on the WEHI-164 target cell line. Incubation of recombinant human TNF and lymphotoxin (LT) with either cathepsin-G or leukocyte elastase resulted in a loss of cytokine bioactivity. Examination of enzyme-treated recombinant cytokines by gel electrophoresis revealed that cathepsin-G cleaved LT into a 12.6-kDa fragment and leukocyte elastase fragmented LT into a 14.1-kDa product. On Western blots cathepsin-G and leukocyte elastase degraded TNF into 11- and 7.6-kDa fragments, respectively. Incubating leukocyte elastase with plasma elastase inhibitor alpha-1-antitrypsin prevented the loss of recombinant TNF bioactivity and blocked the degradation of this cytokine. This study suggests that two of the most abundant neutrophil proteases, cathepsin-G and leukocyte elastase, may be important regulators of TNF and LT bioactivity.     

Biological evaluation of the inhibition of neutrophil elastase by a synthetic beta-lactam derivative

A novel beta-lactam derivative, N-(2-chloromethylphenyl) 3,3-difluoroazetidin-2-one, which behaves as a time-dependent inactivator of leukocyte elastase, has been tested in biological models designed to detect its potential therapeutic value in the treatment of emphysema. Its effect on two types of leukocyte elastase, purified human leukocyte elastase and elastase freshly discharged upon stimulation of guinea pig polymorphonuclear neutrophils, was examined using three methods: the cleavage of a chromogenic peptide substrate, MeO-Suc-Ala-Ala-Pro-Val-NA, the lysis and solubilization of tritiated elastin and the microscopic examination of the damage to lung elastic network. The inhibitor was shown to be effective at preventing proteolysis due to leukocyte elastase. Besides its low cellular toxicity, no apparent hindrance of its efficiency was found in the above quasi in vivo environment. This suggests that this inhibitor may be of potential therapeutic value in elastase-related pathology.     

Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Synthesis and in vitro and in vivo evaluation of the 2-(6'methoxy-3',4'-dihydro-1'-naphtyl)-4H-3,1-benzoxazin-4- one as a new potent substrate inhibitor of human leukocyte elastase.

The title 2-vinyl-4H-3,1-benzoxazin-4-one has been synthesised and tested for inhibitory activity against human leukocyte elastase. The compound has shown activity both in vitro towards human sputum elastase and in vivo in an hemorrhagic assay.     

Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Inhibition of human leukocyte elastase by acetyl and trifluoroacetyl oligopeptide chloromethyl ketones.

The action of human leukocyte elastase on a series of acetyl and trifluoroacetyl tri-, tetra-, and pentapeptide chloromethyl ketones has been investigated. Leukocyte and pancreatic elastases react quite differently with these irreversible inhibitors. For instance, leukocyte elastase has a much lower affinity for the compounds than pancreatic elastase. On the other hand, the inhibition rate constants of the two enzymes are not influenced in the same way by peptide chain elongation. The two elastases, however, share a common property: trifluoroacetyl tri- and tetraalanine chloromethyl ketones are more tightly bound but are less reactive than the corresponding acetylated inhibitors. This behavior is probably due to the formation of nonproductive complexes between the enzymes and the trifluoroacetylated inhibitors.     

Preferential localisation of elastase in cytosol of human myeloid leukemia HL-60 cells.

The subcellular distribution of the elastase in human myeloid leukemia HL-60 cells was studied in comparison with that in normal leukocytes. On differential centrifugation, most of the elastase activity of HL-60 cell lysates was recovered in the 105,000 x g supernatant, while that of human peripheral blood leukocyte lysates was recovered in the 500 x g precipitate (azurophil granule-rich fraction). Moreover, on Percoll density gradient centrifugation, the elastase activity in HL-60 cell extracts was recovered in the lightest fraction with none in the azurophil granule-rich fractions, whereas most of the activity in leukocyte extracts was recovered in the azurophil granule-rich fractions. This subcellular localization of elastase did not change when HL-60 cells differentiated into monocytes and granulocytes by induction with 12-O-tetradecanoyl phorbol-13-acetate and retinoic acid, respectively. Furthermore, on Sephadex G-75 gel filtration, the elastase activity in HL-60 cell extracts was eluted earlier than that in leukocyte extracts. The size estimation indicated that the elastase of HL-60 cells was 36-30 kDa, corresponding to the size of an elastase precursor reported. The relevance of a large form of the elastase in HL-60 cells to its subcellular localization is discussed.     

The degradation of human lung elastin by neutrophil proteinases

Human lung elastin has been isolated by both a degradative and nondegradative procedure and the products obtained found to have amino acid compositions comparable to published results. These elastin preparations, when utilized as substrates for various mammalian proteinases, were solubilized by porcine elastase at a rate six times faster than human leukocyte elastase. Leukocyte cathepsin G also solubilized lung elastin but only at 12% of the rate of the leukocyte elastase. In all cases the elastin prepared by nondegradative techniques proved to be the best substrate in these studies. The differences in the rate of digestion of elastin of the two elastolytic proteinases was readily attributed to the specificity differences of each enzyme as judged by carboxyterminal analysis of solubilized elastin peptides. The plasma proteinase inhibitors, alpha-1-proteinase inhibitor and alpha-2-macroglobulin abolished the elastolytic activity of both leukocyte enzymes, while alpha-1-antichymotrypsin specifically inactivated cathespsin G. Two synthetic inhibitors, Me-O-Suc-Ala-Ala-Pro-Val-CH2Cl (for elastase and Z-Gly-Leu-Phe-CH2Cl (for cathepsin G) were equally effective in abolishing the elastolytic activity of the two neutrophil enzymes. However, inhibition of leukocyte elastase by alpha-1-proteinase inhibitor was significantly suppressed if the enzyme was preincubated with elastin prior to addition of the inhibitor.