化学修饰对胰激肽释放酶稳定性及其性质的影响

以自制高活性ＰＰＫ为材料，在氰基硼氢化钠存在下经水溶性乙醛酸修饰其表面氨基，使基抗不可逆热失活稳定性有显著提高，结果表明，修饰ＰＰＫ在等电点等基本性质方面都有变化，修饰ＰＰＫ的ＢＡＥＥ活性为天然酶的８２％，氨基修饰度为５８％，抗蛋白酶水解，贮藏和冻干稳定性都有加强。     

RFP_（134）对UMR106细胞EGF受体酪氨酸蛋白激酶的调节作用

以大鼠成骨肉瘤细胞（ＵＭＲ１０６）为模型,研究了表皮生长因子（ＥＧＦ）对其受体酪氨酸蛋白激酶（ＴＰＫ）的调节作用。以本实验室从植物中提取纯化的二萜类活性物质（ＲＦＰ１３４）为诱导分化剂,观察了ＲＦＰ１３４对ＵＭＲ１０６细胞ＥＧＦ受体ＴＰＫ的活性和磷酸化作用的影响,并与ＲＡ和ＲＦＰ１３４＋ＲＡ处理细胞做了比较,结果显示ＥＧＦ与其受体结合后能激活ＴＰＫ,使ＴＰＫ活性增加２倍．ＲＦＰ１３４,ＲＡ,ＲＦＰ１３４＋ＲＡ处理细胞后,分别降低ＥＧＦ诱导的受体ＴＰＫ活性５０％,４３％,５５％,降低磷酸化ＴＰＫ含量５５％,３６％,５３％。从结果中发现无ＥＧＦ刺激的细胞也具有受体ＴＰＫ磷酸化作用,用ＲＦＰ１３４,ＲＡ,ＲＦＰ１３４＋ＲＡ处理细胞,分别降低受体磷酸化ＴＰＫ含量５９％,４０％,５７％,而且我们发现用ＥＧＦ诱导的细胞受体ＴＰＫ含量高于无ＥＧＦ作用的细胞．提示ＵＭＲ１０６细胞本身可能具有受体ＴＰＫ活性,能够引起细胞受体自动磷酸化,ＥＧＦ刺激后ＴＰＫ的磷酸化作用增强,可见ＲＦＰ１３４对ＥＧＦ诱导的ＴＰＫ磷酸化和无ＥＧＦ诱导的受体自动磷酸化都具有明显的抑制作用,（并强于ＲＡ）这可能与在第二信使水平上阻抑ＰＴＰＫ活性密切相关     

PEG修饰的辣根过氧化物酶及其在非水介质中的性质

酶的化学修饰可以明显提高酶在有机相中的活力。通过氧化过氧化物酶（ＨＲＰ）的糖链后引入氨基再连接甲氧基聚乙醇（ＰＥＧ）５０００和在酶的肽链上连接ＰＥＧ５０００,发现ＨＲＰ多肽链上修饰后的酶在水相中的活力几乎没有变化,但通过氧化糖链连接ＰＥＧ的酶在水相中的活力下降近２倍。在甲苯及二氧六环含量较高的体系中,修和均呈上升趋势。特别在甲苯体系中两种修饰酶活力都比未经修饰的酶提高了近２倍。稳定性研究表明,不论     

白细胞介素－2的PEG化

用自制的氨基ＰＥＧ化试剂ｒＩＬ－２进行化学修饰,研究了试剂浓度,溶液ｐＨ,反应时间等与ＰＥｃａ－ｒＩＬ－２产率及ＩＬ－２活性保持之间的关系,建立了一套获得稳定修饰度的ＰＥＧ－ｒＩＬ－２的方法。研究发现,反应时间跟修饰度关系不大;溶液ｐＨ对修饰度有一定的影响,中性ｐＨ以上反应都可进行;而试剂浓度直接决定修饰度的高低,过量越多,修饰度越高,而生物活性保留也越低;但低度修饰,对活性几乎没有影响,可保留活性在９５％左右。     

PEG修饰牛血红蛋白的计算机模拟研究

对αβ二聚牛血红蛋白晶体结构的分析和氨基酸钱基溶剂可及表面积的计算表明,牛血红蛋白表面Ｌｙｓ上的氨基适合进行聚乙二醇（ＰＥＧ）修饰,对其修饰不会影响携氧能力,在此基础上设计了连接物连接ＰＥＧ和牛血红蛋白。分子模拟研究结果显示ＰＥＧ修饰牛血红蛋白产物是无免疫原性的。     

PKC、PKA和TPK在血小板激活中的作用

利用~（３２）Ｐ－ＮａＨ_２ＰＯ_４标记猪血小板,然后以ＰＭＡ、凝血酶、ＰＧＥ_１、腺苷等处理,结果表明,随着ＰＭＡ激活ＰＫＣ,血小板发生聚集。３５μｍｏｌ／ＬＰＧＥ_１或１ｍｍｏｌ／ＬｄｂｃＡＭＰ不能抑制５０ｎｍｏｌ／ＬＰＭＡ诱导的血小板聚集,腺苷却能抑制ＰＭＡ诱导的血小板聚集（ＥＣ_（５０）＝０．１ｍｍｏｌ／Ｌ）,ｄｂ－ｃＡＭＰ、腺苷都不能抑制１００ｎｍｏｌ／ＬＰＭＡ诱导的４０ｋＤ蛋白磷酸化。ＰＫＡ激活不能抑制ＰＭＡ激活的ＰＫＣ。在ＰＭＡ、凝血酶激活的血小板中,ＰＫＣ、ＴＰＫ都发生激活,４０ｋＤ底物既是ＰＫＣ的底物又是ＴＰＫ的底物,ＰＫＣ和ＴＰＫ在血小板聚集中起着重要的调节作用。     

蛋白质N端豆蔻酰化修饰的基因工程表达偶联加工系统

对酵母ＮＭＴ基因在大肠杆菌中表达进行较详细的研究,进而构建了复制子为ｐ１５Ａ并含卡那霉素抗性基因的相容性表达质粒ｐＫＺＭＴ,将其与表达质粒ｐＣＺｍＣα１共转化进大肠杆菌ＢＬ２１（ＤＥ３）Ｆ′,进行双质粒表达偶联加工修饰研究,其中ｐＣＺｍＣα１表达底物蛋白小鼠ｃＡＭＰ依赖的蛋白激酶催化亚基α（ＰＫＡ-ｍＣα）。ＳＤＳＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ分析表明,双质粒表达系统中,ＰＫＡ-ｍＣα都得到了稳定的高表达,尤其在２３℃低温诱导表达时,表达产物的可溶性部分明显增多;而酵母ＮＭＴ被控制在有利于活性功能的可溶性低水平表达。［^３Ｈ］ｍｙｒｉｓｔｉｃａｃｉｄ标记测定及放射自显影的结果显示,在大肠杆菌中表达的重组ＰＫＡ-ｍＣα被豆蔻酰化修饰。     

RFP134对UMR106细胞EGF受体酪氨酸蛋白激酶的调节作用

以大鼠成骨肉瘤细胞（ＵＭＲ１０６）为模型,研究了表皮生长因子（ＥＧＦ）对其受体酪氨酸蛋白激酶（ＴＰＫ）的调节作用。以及实验室从植物中提取纯化的二萜类活性物质（ＲＦＰ１３４）为诱导分化剂,观察了ＲＦＰ１３４对ＵＭＰ１０６细胞ＥＧＦ受体ＴＰＫ的活性和磷酸化作用的影响。并与ＲＡ和ＲＦＰ１３４＋ＲＡ处理细胞做了比较。结果显示ＥＧＦ与其受体结合后能激活ＴＰＫ,使ＴＰＫ活性增加２倍。ＲＦＰ１３４,ＲＡ,ＲＦＰ     

Nm23-H1/NDPK-A基因在大肠杆菌中的高效表达及产物纯化的研究

利用聚合酶链反应（ＰＣＲ）技术扩增人二磷酸核苷激酶Ａ亚基（ＮＤＰＫ－Ａ）基因,即ｎｍ２３－Ｈ１／ＮＤＰＫ－Ｋ基因的编码序列,经序列分析后,定向克隆于表达质粒载体ｐＢＶ２２０,在大肠杆菌ＤＨ５α中高效表达出重组人ＮＤＰＫ－Ａ．表达产物为可溶性的非融合蛋白,占菌体总蛋白４２％．斑点ＥＬＩＳＡ法鉴定表明表达产物与ＮＤＰＫ－Ａ标准抗血清呈阳性反应．以ＤＥＡＥ纤维素弱阴离子交换层析、ＣｉｂａｃｒｏｎＢｌｕｅ染料亲和层析结合高效液相排阻色谱技术纯化ｒＮＤＰＫ－Ａ,得纯度为９６．７％的目标蛋白．以反相高效液相色谱法进行酶活性分析,表明纯化的ｒＮＤＰＫ－Ａ能催化ＡＴＰ＋ＵＤＰ＝ＡＤＰ＋ＵＴＰ的反应,比活性为８００Ｕ／ｍｇ蛋白．     

蛋白质定点PEG化研究：97Cys－IFH－γ的巯基专—PEG修饰

蛋白质定点ＰＥＧ化研究：９７Ｃｙｓ－ＩＦＨ－γ的巯基专—ＰＥＧ修饰唐微,常远,徐　飞,郑仲承,刘新垣（中国科学院上海生物化学研究所,２０００３１）关键词人干扰素－γ;定点修饰;半胱氨酸目前进行的ＰＥＧ化反应,多是针对蛋白质肽链上自由末端氨基进行的,由...     

Immobilization of β-galactosidase on modified polypropilene membranes

A new immobilized system: β-galactosidase-modified polypropylene membrane was created. It was obtained 13 different carriers by chemical modification of polypropylene membranes by two stages. The first stage is treatment with K(2)Cr(2)O(7) to receive carboxylic groups on membrane surface. The second stage is treatment with different modified agents ethylendiamine, hexamethylenediamine, hydrazine dihydrochloride, hydroxylamine, o-phenylenediamine, p-phenylenediamine, N,N'-dibenzyl ethylenediamine diacetate to receive amino groups. The quantity of the amino groups, carboxylic groups and the degree of hydrophilicity of unmodified and modified polypropilene membranes were determined. β-Galactosidase was chemically immobilized on the obtained carries by glutaraldehyde. The highest relative activity of immobilized enzyme was recorded at membrane modified with 10% hexamethylenediamine (Membrane 5) - 92.77%. The properties of immobilized β-galactosidase on different modified membranes - pH optimum, temperature optimum, pH stability and thermal stability were investigated and compared with those of free enzyme. The storage stability of all immobilized systems was studied. It was found that the most stable system is immobilized enzyme on Membrane 5. The system has kept 90% of its initial activity at 300th day (pH=6.8; 4°C). The stability of the free and immobilized β-galactosidase on the modified membrane 5 with 10% HMDA in aqueous solutions of alcohols - mono-, diol and triol was studied. The kinetics of enzymatic reaction of free and immobilized β-galactosidase on the modified membrane 5 at 20°C and 40°C and at the optimal pH for both forms of the enzyme were investigated. It was concluded that the modified agent - hexamethylenediamine, with long aliphatic chain ensures the best immobilized β-galactosidase system.     

小分子封闭提高固定化嗜热菌蛋白酶的热稳定性

为了提高固定化嗜热菌蛋白酶的热稳定性,在制备共价固定化嗜热菌蛋白酶的基础上,通过选择氨基酸和醇类小分子来封闭载体表面未反应的活化基团,并考察了固定化酶的催化活性及热稳定性。结果发现：L-Trp和L-Val封闭修饰固定化酶时,在80℃的水浴中加热150 min后其剩余活力仍为93.4%和98.6%,其效果约为未经小分子封闭的固定化嗜热菌蛋白酶的2倍。所筛选的几种小分子物质中,叔戊醇、L-Trp、L-Val及L-Ala不仅能提高固定化嗜热菌蛋白酶的热稳定性,而且也可以提高固定化酶的相对活力,从而更有利于其在工业生产中的应用。     

棕榈酰化超氧化物歧化酶的制备及性质研究

为了增强超氧化物歧化酶的稳定性,用棕榈酸对其进行了修饰,在修饰条件下,酶分子表面氨基修饰率为55％时,酶的活力回收为63％。修饰后的酶在耐热、耐酸、耐碱、抗有机溶剂变性和抗蛋白水解能力上均高于天然超氧化物歧化酶,为将超氧化物歧化酶作成实用药物和进一步扩大其应用范围创造了条件。     

Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.     

Surface activity and molecular characteristics of cuttlefish skin gelatin modified by oxidized linoleic acid

Surface activity and molecular changes of cuttlefish skin gelatin modified with oxidized linoleic acid (OLA) prepared at 60, 70 and 80 °C at different times were investigated. Modification of gelatin with OLA could improve surface activity of resulting gelatin as evidenced by the decreased surface tension and the increased foaming and emulsifying properties. Interaction between OLA and gelatin led to the generation of carbonyl groups, loss of free amino content and the increase in particle size of resulting gelatin. Emulsion stabilized by modified gelatin had the smaller mean particle diameter with higher stability, compared with that stabilized by gelatin without modification.     

多聚磷酸激酶及其在ATP再生体系构建中的进展

构建高效的腺嘌呤核苷三磷酸(adenosinetriphosphate,ATP)再生体系可显著提高生物催化磷酸基团转移反应的效率。多聚磷酸激酶(poly phosphate kinase, PPK)能利用来源广、廉价且稳定的多聚磷酸(polyphosphate, Poly P)盐作为磷酸基供体，能够实现单磷酸腺苷(adenosine monophosphate,AMP)、二磷酸腺苷(adenosinediphosphate,ADP)、ATP、PolyP之间磷酸基的高效定向转移，已成为构建ATP再生体系的首选。本文介绍了不同类型PPK的结构特征、相关催化机制以及不同来源的PPK在酶活、催化效率、稳定性和底物偏好性的特征差异；归纳和列举了针对野生PPK酶学性质不足进行分子改造的实例，并对PPK在ATP再生体系构建的研究进展进行了总结。     

Chemical modification of lysine residues of beta-1,3-glucanase from Spisula sachalinensis

The effects of chemical modification of the amino groups of lysine residues on the activity of beta-1.3-glucanase from Spisula sachalinensis were studied. Modification of two lysine residues per molecule did not affect either the enzyme activity with respect to laminarine, nor the Km value. The modified beta-1.3-glucanase retains the ability to catalyze the transglycosylation and cleaves the high molecular weight CM-pachyman at the same rate as does the native enzyme. No significant changes in the enzyme thermal stability were observed. Thus, the modified enzyme groups cannot be involved in the enzyme active center and are exposed on the surface of the protein globule. The chemical modification was shown to have no effect on the enzyme kinetics, which is essential for its immobilization.     

Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like beta-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg(2+), and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.     

Identification of "buried" lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors.

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or "buried" amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is "buried" in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.     

Changes in phosphofructokinase activity upon exposure of muscles and muscle extracts to injurious agents

A study has been made on changes of outflux, extractability and activity of phosphofructokinase (PPK) under the action of heating, and of urea on the frog's skeletal muscles and on their extracts. Under the action of heating on muscles, the decrease of PPK activity (to 35%) is first revealed 34--36 degress C, when decrease of excitability and the contracture are not yet detected, and the extractability of the total water-soluble protein does not change. At the start of contracture, and at the decrease of excitability (at 38 degrees C) PPK in the muscle loses its activity. The thermolability of PPK is the greatest one compared to all the enzymes investigated before. The data on the high thermolability of PPK are discussed in terms of the regulating role of PPK in glycolysis. The PPK activity of extracts of muscles altered by urea, during the action not accompanied by the death of muscles, does not change. At the irreversible disappearance of muscle excitability, PPK is inactivated. PPK in the cell is more stable to injuring agents than PPK in isolated state.