弹性蛋白酶的研究(二) 右旋糖酐对弹性蛋白酶的化学修饰及其某些性质的研究

本文用溴化氰活化右旋糖酐,对弹性蛋白酶进行共价修饰,以便改变其若干性质,使之更有利于应用。实验结果表明,修饰酶不仅完全保留了天然酶的活性和抗胰蛋白酶水解能力,而且修饰酶在耐热性、耐酸性和抗胃蛋白酶水解能力上都明显地优于天然酶。修饰酶较天然酶稳定,具有较高的应用价值。     

PEG2修饰^125I—L—天冬酰胺酶在小鼠体内的分布与代谢

本文对ＰＧＦ２修饰＾１２５Ｉ－Ｌ天冬酰胺酶在小鼠体内的分布与代谢进行了研究，结果表明：ＰＥＧ２修饰Ｌ－天冬酰胺酶在小鼠体内半衰期与天然酶相比明显延长，修饰酶是５４ｈ，天然酶是４ｈ，前者是后者的１３．５部，修饰酶与天然酶在体内分布与代谢也明显不同，修饰酶主要分布在肝脏，并在肝脏代谢，而天然酶主要分布在肾脏，并通过肾脏代谢，修饰酶没有体内积留现象，这些结果暗示，在治疗白血病上修饰酶较天然酶更有效。     

右旋糖苷对猪血超氧化物歧化酶的共价修饰及性质研究

用高碘酸钠活化右旋糖苷对猪血SOD进行共价修饰,修饰SOD用Sephadex G-150测其分子量约为130,000;修饰酶保留了天然酶的紫外及萤光吸收特性;修饰酶较未修饰酶的热稳定性、PH稳定性,以及对尿素、EDTA的稳定性都有较显著的提高。     

修饰SOD—CTS复合酶的制备及其性质研究

报道了SOD-CTS复合酶的化学修饰。药用淀粉用高碘酸钠氧化后,与复合酶共价结合制得了修饰酶。与天然酶相比,修饰酶对温度、酸、碱有较强的抗性,且具有较强的抗蛋白酶水解能力,     

天然药物化学成分结构修饰研究进展

对近年来的天然药物化学成分结构修饰相关文献进行归纳、分析和整理,主要从多糖、黄酮类、香豆素类、生物碱类等化合物结构修饰方面进行了总结。结果发现天然药物化学成分结构修饰或许可以为天然药物的进一步合理应用以及相关新药的开发开辟出一条新的重要途径。天然药物具有广泛的生物活性,潜在开发应用价值巨大。     

蚯蚓纤溶酶化学修饰的研究

以三聚氰氯为活化剂，采用活化聚乙二醇(PEG)对环毛蚯蚓纤溶酶进行化学修饰，优化了修饰条件，得到了PEG-EFE的加合物(修饰酶)，并研究了PEG-EFE的部分酶学性质。修饰酶的活力为天然酶的85％，结果表明酶的活性中心基本保持不变。对修饰酶的残留氨基测定表明，70％的可滴定氨基参加了反应，且EFE的活性改变较小，可以推测PEG是连接在蛋白质表面上的。     

组蛋白去乙酰化酶抑制剂治疗血液肿瘤研究进展

表观遗传学修饰的改变,可使基因表达异常,并促进肿瘤的发生发展.表观遗传学修饰包括DNA甲基化修饰及组蛋白修饰.由组蛋白乙酰基转移酶及组蛋白去乙酰化酶调控的乙酰化修饰,是最重要的组蛋白修饰.应用组蛋白去乙酰化酶抑制刺对其进行调控,可使肿瘤细胞生长受抑,发生凋亡,并可增强肿瘤对化疗的敏感性.单独应用组蛋白去乙酰化酶抑制剂或联合其他化疗药物,成为抗血液肿瘤研究与治疗的新方向.     

肽和蛋白质类药物的给药系统(Ⅱ)

<正>3 蛋白质─聚合物共轭体 重组蛋白因缺乏糖基而使其溶解性和稳定性较差,并且有抗原性,与以聚乙二醇（PEG）为代表的水溶性聚合物通过共价键形成轭合物后,轭合物的性质较之未经修饰的原物质有很大改善,溶解度提高,稳定性增强,生物半衰期延长,毒副作用（免疫原性和过敏反应）降低甚至消除,疗效提高,因此在临床上得到应用。 白细胞介素-2经PEG修饰后的轭合物以适当的剂量和方式给药,可以延长血液中IL-2活性达到最大值的时间,延长循环寿命,减轻免疫原性,提高治疗指数和疗效。PEG-天冬酰胺酶轭合物的半衰期较之天然酶延长了17倍,免疫原性也有所降低,治疗指数得以提高。用PEG修饰,在临床上得到应用的酶还有:SOD（超氧化物歧化酶）、ADA（腺苷脱氨酶）、尿酸酶等,都表现出相似的特性。     

SOD特性改善技术研究进展

超氧化物歧化酶（SOD）的发现。为氧代谢的研究开创了新的局面。氧自由基被认为是机体重要的信号分子,参与了多种信号途径,与多种疾病的发生发展相关。SOD作为机体最重要的抗氧化酶体之一,直接清除机体产生的超氧自由基,阻止了机体的过氧化。但自然存在的SOD在体内代谢快。且分子量大,不易透过细胞膜而进入靶细胞,限制了其在机体内的应用。针对天然SOD的不足,早期国内外学者通过化学方法对其进行修饰研究,得到了多种修饰酶,为其在多领域的应用奠定了基础。近年来也有不少学者利用固定化酶技术、基因工程等手段对其进行特性改造。使其跨膜性、靶向性大大提升。成果显著。本文试图对国内外超氧化物歧化酶功能特性改善技术的研究进行概括。     

磺化在天然产物结构修饰中的应用

磺化是一种重要的结构修饰手段，可以改善化合物的水溶性，并可能增加其生物活性，综述磺化在二萜醌类、酚类、黄酮类以及多糖四类天然产物结构修饰中的应用。     

几种因素在L-天冬酰胺酶化学修饰中对酶活力及抗原性的影响

为了寻求在较好地保持酶活力的同时解除L-天冬酰胺酶抗原性的方法,采用不同分子量的乙酸酐、右旋糖酐和单甲氧基聚乙二醇,作为修饰剂和不同的修饰方法对该酶进行了化学修饰。结果表明在保持酶活性和降低抗原性方面,大分子修饰剂右旋糖酐、单甲氧基聚乙二醇优于小分子乙酸酐,底物保护修饰优于直接修饰;活化PEG,优于活化PEG₁。在底物保护下的PEG,修饰酶其抗原性完全解除的同时,酶活力保持在30%以上。     

Effects of modification of tyrosines 3 and 62 (63) on enzymatic and toxicological properties of phospholipases A2 from Naja nigricollis and Naja naja atra snake venoms

Previously we selectively modified His (48), Arg, Lys, Asp, Glu and Trp residues in the basic phospholipase A2 from Naja nigricollis and the acidic phospholipase A2 from N. n. atra snake venoms. Evidence was obtained for the existence of separate but perhaps overlapping sites responsible, respectively, for their enzymatic and pharmacological properties. We have now modified one or two (Tyr 3, Tyr 62 [63], Tyr 3 + 62 [63]) out of the nine tyrosine residues in these enzymes using p-nitrobenzenesulfonyl fluoride. The derivatives were separated by HPLC, and modified residues determined by amino acid analysis. Enzymatic activity was tested on lecithin--Triton mixed micelles, egg yolk and heart and diaphragm homogenates. The N. nigricollis modified derivatives retained a greater percentage of their enzymatic activities than did the N. n. atra derivatives and also a greater percentage of their activity on natural substrates than on lecithin--Triton mixed micelles. The greatest loss in activity resulted when both tyrosines were modified and the least when tyrosine 3 was modified. Modification of tyrosine 62 of N. nigricollis caused a much greater loss of intraventricular lethal potency than of enzymatic activity, whereas modification of tyrosine 3 of N. n. atra increased lethal potency over six-fold while enzymatic activity decreased about 60%. Examples of dissociation between enzymatic and pharmacological potencies were also noted when hemolytic, anticoagulant and cardiotoxicity on isolated ventricular muscle were measured. The extents of phospholipid hydrolysis were relatively low in brain homogenates, synaptic plasma membranes and heart ventricular muscle. However, they were similar for the native enzymes and all of the tyrosine modified derivatives. These tyrosines do not appear to be part of the enzymatic active site, even though they are thought to be associated with substrate and calcium binding. These results strengthen our earlier conclusion that some pharmacological effects of phospholipase A2 are not due to enzymatic hydrolysis, and that there are separate but perhaps partly overlapping sites for enzymatic and pharmacological activities.     

Synthesis and Inverse Emulsion Polymerization of Aminated Acrylamidodextran

Abstract— A chemically modified form of dextran was prepared, having a polymerizable moiety (acrylamide) and a reactive functional group (primary amine). Dextran was activated with 4-nitrophenyl-chloroformate (24 mol per polysaccharide, 9·8 mol per 100 glucose residues); 9·8% glucose residues were converted to aliphatic carbonates and 5·2% were converted to cyclic carbonates. The activated dextran was coupled with trityldiaminoethane (8 mol per 100 glucose residues), reactivated with 4-nitrophenylchloro-formate, then coupled with acryloamidodiaminohexane (6·8 mol per 100 glucose residues). The trityl group was removed by hydrolysis with trifluoroacetic acid to yield the required aminated acryloamidodextran. The modified dextran was shown to be polymerizable by inverse emulsion polymerization. Submicron particles were successfully prepared, containing functional amine groups suitable for preparing drug conjugates or for modifying the surface properties of this biomaterial.     

Effect of carboxylate group modification on enzymatic and cardiotoxic properties of snake venom phospholipases A2

By treating Naja nigricollis and Naja naja atra phospholipase A₂ with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity ($\text{1}\text{4}$1 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Caboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12 – 17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17 – 33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Gluresidues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A₂ are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

Inhibition of neutrophil elastase activity by phenolic compounds from plants.

The influence of 40 phenolic compounds from plants were tested on the enzymatic activity of neutrophil elastase (EC 3.4.21.37). Among the flavonoids especially the compounds with a catecholic structure showed a strong inhibitory activity in the range of mumol/l, hyperosid was the strongest inhibitor with an IC50 of about 0.3 mumol/l. Lipophilic caffeic acid esters and phenolic compounds with catecholic structure elements in general inhibited the enzyme. Because of the specific role of neutrophil elastase in the inflammatory process its inhibition by phenolic natural compounds might be one explanation for the use of medicinal plants containing phenolic compounds in the treatment of inflammation.     

Inhibition of neutrophil elastase and thrombin activity by caffeic acid esters

Natural and synthetic caffeic acid esters were assayed for their enzymatic activity versus neutrophil elastase (EC 3.4.21.37) and thrombin (EC 3.4.21.5). Lipophilic caffeic acid esters inhibited neutrophil elastase activity and the inhibition rate was enhanced with increasing length of the aliphatic chain of the alcohol component. The geometry of the chain seems to be more important than the number of carbon atoms. The most inhibitory compound was n-octylcaffeic acid ester with an IC50 value of 1.0 microM. Thrombin activity was only weakly inhibited by the caffeic acid esters thus demonstrating a specificity for neutrophil elastase. Because of its critical role in inflammatory processes, inhibition of neutrophil elastase by caffeic acid esters might be of importance in the treatment of inflammation.     

水蛭提取方法及生理活性的研究

目的筛选较优的水蛭提取方法。方法采用原粉匀浆、水煎煮、水煎醇沉、胃蛋白酶酶解、胰蛋白酶酶解、仿生酶解等不同的方式提取水蛭,考察各提取方法的得膏率、对凝血酶原时间、纤溶活性、大鼠凝血时间及静脉血栓重量的影响。结果各项指标中以上六种工艺与空白对照组相比较均有显著性差异(P<0.05);各酶解工艺组与空白对照组相比有极显著性差异(P<0.01),其中仿生酶解液组与匀浆液组相比有极显著性差异(P<0.01),仿生酶解组明显优于其他酶解组,同时仿生酶解的得膏率最高。结论仿生酶解方法所得样品液的得膏率及生理活性作用较其他提取方法有极大提高,生物利用度更高,值得推广应用。     

Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja naja atra snake venoms

Modification of one histidine residue at the active site in the phospholipases A₂ from N. nigricollis, H. haemachatus and N. naja atra snake venoms reduced the enzymatic activity to 0·–0·8% that of the native species, while the Km values for the first two enzymes listed remained practically unchanged. Also unchanged following modification were the characteristic substrate specificities of the three enzymes. While it appears most likely that the residual activity of the modified enzymes originates from contamination by non-modified species, the modified enzyme might have residual activity of its own. The decrease in enzymatic activity was accompanied by a decrease in all other biochemical and physiological parameters studied such as: direct hemolysis (for N. nigricollis phospholipase), ability to hydrolyze red cell phospholipids, intravenous ld₅₀ in mice, intraventricular ld₅₀ and cd₅₀ in rats, brain phospholipid hydrolysis and ability to block action potentials in eel electroplax. The results support the view that catalytic activity is essential for toxic action.     

Chemical modification of hyaluronidase regulates its inhibition by heparin.

Chemical modification of surface amino groups of bovine testicular hyaluronidase with aldehyde dextran was conducted. It was found that with the increase of modification degree of hyaluronidase amino groups the value of residual enzymatic catalytic activity is decreased rather monotonously. It turned out that the value of inhibition of enzyme activity by heparin considerably depends on modification degree of enzyme. This dependence is of a threshold character. Sharp conformational changes in the enzyme occurring at 70-90% degree of its modification considerably lowers heparin inhibition. The higher the degree of hyaluronidase modification, the weaker its inhibition by heparin. More completely/deeply modified derivatives of hyaluronidase (modification degree 96-100%) are practically not inhibited by heparin. Thus, chemical conjugation of hyaluronidase with aldehyde dextran regulates the value of enzyme inhibition by heparin. Hyaluronidase modification becomes an informative tool to study the mechanism of inhibition of its enzyme activity and an efficient means for the development of new therapeutic preparations improving tissue permeability during cardiovascular injuries.     

Effects of heparin and related molecules upon neutrophil aggregation and elastase release in vitro

1 Neutrophil-derived elastase is an enzyme implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Heparin inhibits the enzymatic activity of elastase and here we provide evidence for the first time that heparin can inhibit the release of elastase from human neutrophils. 2 Unfractionated and low molecular weight heparins (UH and LMWH, 0.01-1000 U ml(-1)) and corresponding concentrations (0.06-6000 micro g ml(-1)) of nonanticoagulant O-desulphated heparin (ODH), dextran sulphate (DS) and nonsulphated poly-L-glutamic acid (PGA) were compared for their effects on both elastase release from and aggregation of neutrophils. 3 UH, ODH and LMWH inhibited (P<0.05) the homotypic aggregation of neutrophils, in response to both N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-6) M) and platelet-activating factor (PAF, 10(-6) M), as well as elastase release in response to these stimuli, in the absence and presence of the priming agent tumour necrosis factor-alpha (TNF-alpha, 100 U ml(-1)). 4 DS inhibited elastase release under all the conditions of cellular activation tested (P<0.05) but had no effect on aggregation. PGA lacked efficacy in either assay, suggesting general sulphation to be important in both effects of heparin on neutrophil function and specific patterns of sulphation to be required for inhibition of aggregation. 5 Further investigation of the structural requirements for inhibition of elastase release confirmed the nonsulphated GAG hyaluronic acid and neutral dextran, respectively, to be without effect, whereas the IP(3) receptor antagonist 2-aminoethoxydiphenylborate (2-APB) mimicked the effects of heparin, itself an established IP(3) receptor antagonist, suggesting this to be a possible mechanism of action.