Novel serine proteases encoded by two cytotoxic T lymphocyte-specific genes

Genes that are expressed exclusively in cytotoxic T cells should encode proteins that are essential for target cell lysis in cell-mediated immune responses. The sequences of two cytotoxic T lymphocyte-specific complementary DNA's (cDNA's) suggest that the two genes encode serine proteases. A full-length cDNA corresponding to one of the genes was isolated and sequenced. The predicted protein resembles serine proteases in that it includes all the residues that form the catalytic triad of the active site of serine proteases. Moreover, it has sequence characteristics thought to occur only in rat mast cell protease type II. These results are in accord with the view that a protease cascade plays a key role in cytotoxic T-cell activation.     

丝氨酸蛋白酶研究进展

综述了丝氨酸蛋白酶的结构与功能、蛋白质工程、表达系统和应用。丝氨酸蛋白酶是一类以丝氨酸为活性中心的重要的蛋白水解酶,在生物有机体中发挥着重要而又广泛的生理作用,具有广泛的研究和应用价值。它们的活性部位都含Ser、His、Asp,并具有相同的催化机制。丝氨酸蛋白酶超家族成员执行多种多样的生理功能,在很多致病过程以及细胞内的信号转导等方面起着重要作用。正是由于丝氨酸蛋白酶在结构上的微小变化,从而引起了其在功能上的进化。     

Fluctuations and bistabilities on catalyst nanoparticles

We show that coverage fluctuations on catalyst particles can drastically alter their macroscopic catalytic behavior. Scrutinizing the occurrence of kinetic bistabilities, it is demonstrated by molecular beam experiments on model catalysts that macroscopically observable bistabilities vanish completely with decreasing particle size, as previously predicted by theory. The effect is attributed to fluctuation-induced transitions between two kinetic reaction regimes, with a transition rate controlled by both particle size and surface defects. These results suggest that fluctuation-induced effects represent a general phenomenon affecting the reaction kinetics on nanostructured surfaces.     

Engineering enzyme specificity by "substrate-assisted catalysis"

A novel approach to engineering enzyme specificity is presented in which a catalytic group from an enzyme is first removed by site-directed mutagenesis causing inactivation. Activity is then partially restored by substrates containing the missing catalytic functional group. Replacement of the catalytic His with Ala in the Bacillus amyloliquefaciens subtilisin gene (the mutant is designated His64Ala) by site-directed mutagenesis reduces the catalytic efficiency (kcat/Km) by a factor of a million when assayed with N-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide (sFAAF-pNA). Model building studies showed that a His side chain at the P2 position of a substrate bound at the active site of subtilisin could be virtually superimposed on the catalytic His side chain of this serine protease. Accordingly, the His64Ala mutant hydrolyzes a His P2 substrate (sFAHF-pNA) up to 400 times faster than a homologous Ala P2 or Gln P2 substrate (sFAAF-pNA or sFAQF-pNA) at pH 8.0. In contrast, the wild-type enzyme hydrolyzes these three substrates with similar catalytic efficiencies. Additional data from substrate-dependent pH profiles and hydrolysis of large polypeptides indicate that the His64Ala mutant enzyme can recover partially the function of the lost catalytic histidine from a His P2 side chain on the substrate. Such "substrate-assisted catalysis" provides a new basis for engineering enzymes with very narrow and potentially useful substrate specificities. These studies also suggest a possible functional intermediate in the evolution of the catalytic triad of serine proteases.     

The structural basis of ribosome activity in peptide bond synthesis

Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin. The unusual pK(a) (where K(a) is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.     

An unexpectedly efficient catalytic antibody operating by ping-pong and induced fit mechanisms

A transition state analogue was used to produce a mouse antibody that catalyzes transesterification in water. The antibody behaves as a highly efficient catalyst with a covalent intermediate and the characteristic of induced fit. While some features of the catalytic pathway were programmed when the hapten was designed and reflect favorable substrate-antibody interactions, other features are a manifestation of the chemical potential of antibody diversity. The fact that antibodies recapitulate mechanisms and pathways previously thought to be a characteristic of highly evolved enzymes suggests that once an appropriate binding cavity is achieved, reaction pathways commensurate with the intrinsic chemical potential of proteins ensue.     

Bacterial resistance to beta-lactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 A resolution

beta-lactamases are enzymes that protect bacteria from the lethal effects of beta-lactam antibiotics, and are therefore of considerable clinical importance. The crystal structure of beta-lactamase from the Gram-positive bacterium Staphylococcus aureus PC1 has been determined at 2.5 angstrom resolution. It reveals a molecule of novel topology, made up of two closely associated domains. The active site is located at the interface between the domains, with the key catalytic residue Ser70 at the amino terminus of a buried helix. Examination of the disposition of the functionally important residues within the active site depression leads to a model for the binding of a substrate and a functional analogy to the serine proteases. The unusual topology of the secondary structure units is relevant to questions concerning the evolutionary relation to the beta-lactam target enzymes of the bacterial cell wall.     

A portrait of Alzheimer secretases--new features and familiar faces

The amyloid beta-peptide (Abeta) is a principal component of the cerebral plaques found in the brains of patients with Alzeheimer's disease (AD). This insoluble 40- to 42-amino acid peptide is formed by the cleavage of the Abeta precursor protein (APP). The three proteases that cleave APP, alpha-, beta-, and gamma-secretases, have been implicated in the etiology of AD. beta-Secretase is a membrane-anchored protein with clear homology to soluble aspartyl proteases, and alpha-secretase displays characteristics of certain membrane-tethered metalloproteases. gamma-Secretase is apparently an oligomeric complex that includes the presenilins, which may be the catalytic component of this protease. Identification of the alpha-, beta-, and gamma-secretases provides potential targets for designing new drugs to treat AD.     

Mn2+催化臭氧化去除腐殖酸的试验研究

以天然水体中常见的金属离子Mn2+为催化剂,研究了其对水中溶解性腐殖酸的催化臭氧化效能.结果表明,Mn2+对臭氧化去除腐殖酸有明显的催化作用,反应25 min,对腐殖酸的去除率可达82.3%,较单独臭氧化提高了24.3%;通过Na2CO3对催化反应的影响验证了Mn2+催化臭氧化反应遵循自由基反应原理;同时也发现催化效率随着臭氧流量的增大而提高,随着pH值的升高而提高;且少量Mn2+投加即可产生明显的催化作用,过量则会产生抑制作用.     

The dynamic energy landscape of dihydrofolate reductase catalysis

We used nuclear magnetic resonance relaxation dispersion to characterize higher energy conformational substates of Escherichia coli dihydrofolate reductase. Each intermediate in the catalytic cycle samples low-lying excited states whose conformations resemble the ground-state structures of preceding and following intermediates. Substrate and cofactor exchange occurs through these excited substates. The maximum hydride transfer and steady-state turnover rates are governed by the dynamics of transitions between ground and excited states of the intermediates. Thus, the modulation of the energy landscape by the bound ligands funnels the enzyme through its reaction cycle along a preferred kinetic path.     

家蚕丝氨酸蛋白酶BmSP141的表达特征及对饥饿的响应

【目的】分析家蚕(Bombyx mori)丝氨酸蛋白酶基因BmSP141序列信息,明确其时空表达模式,结合饥饿与重新喂食处理对其表达的影响,探究该基因在家蚕中的功能。【方法】对家蚕丝氨酸蛋白酶基因BmSP141进行T-A克隆,得到其编码区核苷酸序列;应用生物信息学在线网站对该基因编码区推导的氨基酸序列、分子量、结构域等信息进行分析;利用ClustalX1.8和MEGA5.02软件对BmSP141与其他物种的丝氨酸蛋白酶进行多序列比对和系统发生树分析;构建原核表达载体p28-BmSP141,并转化到Rosetta（DE3）菌株中诱导表达,利用镍柱亲和层析纯化重组蛋白。利用半定量RT-PCR和实时荧光定量PCR方法对其组织和时期表达特征进行分析;采用蛋白质免疫印迹（Western blot）技术,分别分析BmSP141蛋白在家蚕5龄第3天各组织的表达情况和5龄幼虫中肠的表达变化;通过免疫荧光定位分析BmSP141蛋白在4龄幼虫中肠的分布情况;利用实时荧光定量PCR和Western blot方法对饥饿处理和再喂食后BmSP141的表达情况进行分析。【结果】BmSP141编码含有292个氨基酸的蛋白,其中第1-17位氨基酸为信号肽,其成熟体蛋白预测分子量为25.9 kD,等电点为7.8。同源序列比对表明,BmSP141与烟芽夜蛾丝氨酸蛋白酶和蓓带夜蛾丝氨酸蛋白酶序列同源性较高,分别达到62%和63%。这些同源序列具有保守的催化三联体,与活性相关的基序也很保守,但与胰凝乳蛋白酶保守的底物特异性位点相比,BmSP141的底物特异性位点发生改变。在16和37℃条件下诱导后,重组蛋白均以包涵体形式表达,经镍柱亲和层析纯化后得到了较纯的重组蛋白,并用该蛋白制备了效价较高的多克隆抗体。组织和时期表达特征分析表明,该基因主要在家蚕幼虫的中肠组织特异性表达, 且在幼虫起蚕到食桑期表达逐渐增加,但眠期表达下降,蛹期和成虫期表达水平较低。Western blot分析也表明,BmSP141蛋白仅在家蚕中肠组织表达,且从5龄起蚕到上蔟表达量先增加后降低。免疫荧光定位结果表明,BmSP141定位于中肠上皮细胞的细胞质中,进一步证实BmSP141在中肠特异表达。在转录和翻译水平,BmSP141饥饿处理后表达量显著下调,而重新喂食后其表达量上调,表明BmSP141的表达受到消化道食物的诱导。【结论】家蚕丝氨酸蛋白酶BmSP141在中肠组织表达,在幼虫期食桑期高表达,蛹期和成虫期表达水平较低,受消化道食物的诱导而上调表达,推测该基因可能参与家蚕中肠的消化过程。     

Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody

Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.     

中药蒲黄纤溶蛋白的分离纯化及部分性质

用DEAE Sepharose Fast Flow离子交换层析从中草药蒲黄中分离纯化一种纤维蛋白溶解酶,用纤维蛋白板检测分离后组分的纤溶蛋白活性,天然电泳检测分离效果,分离后得到了比活力为25.29的单一组分,SDS电泳确定该组分的表观分子量为31 000 D,同时比较了不同蛋白酶抑制剂对该组分的抑制作用。结果表明:该组分的纤维蛋白水解酶活性被PMSF、亮抑肽素、抑肽酶、大豆胰蛋白酶抑制剂以及苯甲脒强烈抑制,表明该粗提物中的蛋白酶属于丝氨酸蛋白酶家族。     

野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析(英文)

[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.     

大蒜秸秆的热解及其催化剂研究

针对大蒜秸秆难利用的问题,对其进行了热解研究,发现生物质内的无机盐确实可以起到热解催化的作用.同时研究了没有催化剂和有催化剂两种情况下的热解行为,发现无催化剂时大蒜秸秆在500 ～600℃间失重百分比最大,且裂解产油率随着温度升高而增加,在550℃时产油率最高,随后随着温度增加,二次裂解加剧,产油率降低;而在KOH的催化作用下产油率明显增加,产油率几乎增加了15％,但产油率峰值仍在500 ～600℃.并发现KOH促进大蒜秸秆表面皮下细胞及皮下纤维层进行分解是主要的作用.     

野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析

[目的]构建野桑蚕中肠组织cDNA文库并分离其丝氨酸蛋白酶基因。[方法]利用TaKaRa公司生产的cDNA Library Construction Kit构建了野桑蚕中肠组织的cDNA文库,并采用测序法克隆分析丝氨酸蛋白酶基因cDNA。[结果]经鉴定,文库的滴度达6.2×105pfu/ml,文库插入片段的平均大小为1.2 kb左右。从文库的测序结果中获得了野桑蚕丝氨酸蛋白酶基因片段(登录号:EU672968),序列分析结果显示:该基因片段由854个核苷酸组成,编码284个氨基酸残基。通过对该基因片段编码的氨基酸和其他10种昆虫的丝氨酸蛋白酶基因编码的氨基酸的同源性分析,发现该氨基酸序列与其他丝氨酸蛋白酶具有一定的同源性。[结论]该基因的发现对于研究家蚕和野桑蚕对外来入侵物的抗性具有重要意义。     

Evolution of shape complementarity and catalytic efficiency from a primordial antibody template

The crystal structure of an efficient Diels-Alder antibody catalyst at 1.9 angstrom resolution reveals almost perfect shape complementarity with its transition state analog. Comparison with highly related progesterone and Diels-Alderase antibodies that arose from the same primordial germ line template shows the relatively subtle mutational steps that were able to evolve both structural complementarity and catalytic efficiency.     

催化动力学电位法测定海产品中微量碘

[目的]建立一种测定海产品中微量碘的催化动力学电位法。[方法]采用单因素试验考察了催化动力学电位法测定海产品中微量碘的影响因素,并测定了海带（Laminaria japonica）、紫菜（Porphyra teneraKjellman）和海虾中碘的含量。[结果]建立的催化动力学电位法测定碘含量的线性范围为8-28 ng/ml,方法的检出限为5.2 ng/ml,回收率大于97.0%,相对标准偏差（RSD）小于1.1%,方法准确可靠。采用该法测定的海带、紫菜和海虾中碘的含量分别为31.98、27.38、18.40μg/g,RSD分别为1.48%、0.70%、1.61%。[结论]该研究建立的方法不需要避光,操作简单,温度控制方便,用于海产品中微量碘的测定结果令人满意。     

大肠杆菌外膜蛋白酶基因OmpT的克隆及表达

OmpT(Outer-membrane proteases T)是革兰氏阴性细菌分泌到细胞表面具有丝氨酸蛋白酶活性的一种重要蛋白。利用大肠杆菌OmpT降解抗菌肽特性来筛选和改造新型抗菌肽,可以为开发抗OmpT蛋白酶水解新抗菌肽序列提供新的思路和创造条件。根据大肠杆菌外膜蛋白酶OmpT的基因序列设计一对引物,应用聚合酶链式反应(PCR)方法,从大肠杆菌K12基因组中扩增获得一段为954 bp的序列,测序结果显示此序列与已公布序列同源性达99.99%。将其序列定向克隆到原核表达载体pET28a上构建重组表达质粒pET28a-OmpT。经IPTG诱导后,在表达宿主菌中特异性的表达出分子量约为36 kDa且具有生物活性的OmpT蛋白。生长曲线试验显示,抗菌肽LL37对带重组表达质粒pET28a-OmpT的大肠杆菌生长没有影响,而对照组的生长明显受到抗菌肽的抑制作用。     

纳米金催化剂Au/Al_2O_3的制备及其催化活性研究

用吸附色谱柱法制备了Au/Al2O3催化剂,分析了金粒径大小、酸度、液固比、载体等因素对纳米金催化剂Au/Al2O3活性的影响.结果表明：当粒径为2～3 nm,pH为9,液固比为4 1时,催化剂Au/Al2O3的活性最高.