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51.
Zhang K Zang TZ Yang W Sun Y Mu Y Liu JQ Shen JC Luo GM 《The Journal of biological chemistry》2006,281(18):12516-12520
Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency. 相似文献
52.
Luo N Wang X Chung BH Lee MH Klein RL Garvey WT Fu Y 《American journal of physiology. Endocrinology and metabolism》2011,301(1):E180-E186
Epidemiological studies have associated low circulating levels of the adipokine adiponectin with multiple metabolic disorders, including metabolic syndrome, obesity, insulin resistance, type II diabetes, and cardiovascular disease. Recently, we reported that adiponectin selectively overexpressed in mouse macrophages can improve insulin sensitivity and protect against inflammation and atherosclerosis. To further investigate the role of adiponectin and macrophages on lipid and lipometabolism in vivo, we engineered the expression of adiponectin in mouse macrophages (Ad-TG mice) and examined effects on plasma lipoproteins and on the expression levels of genes involved in lipoprotein metabolism in tissues. Compared with the wild-type (WT) mice, Ad-TG mice exhibited significantly lower levels of plasma total cholesterol (-21%, P < 0.05) due to significantly decreased LDL (-34%, P < 0.05) and VLDL (-32%, P < 0.05) cholesterol concentrations together with a significant increase in HDL cholesterol (+41%, P < 0.05). Further studies investigating potential mechanisms responsible for the change in lipoprotein cholesterol profile revealed that adiponectin-producing macrophages altered expression of key genes in liver tissue, including apoA1, apoB, apoE, the LDL receptor, (P < 0.05), and ATP-binding cassette G1 (P < 0.01). In addition, Ad-TG mice also exhibited higher total and high-molecular-weight adipnection levels in plasma and increased expression of the anti-inflammatory cytokine IL-10 as well as a decrease in the proinflammatory cytokine IL-6 in adipose tissue. These results indicate that macrophages engineered to produce adiponectin can influence in vivo gene expression in adipose tissue in a manner that reduces inflammation and macrophage infiltration and in liver tissue in a manner that alters the circulating lipoprotein profile, resulting in a decrease in VLDL and LDL and an increase in HDL cholesterol. The data support further study addressing the use of genetically manipulated macrophages as a novel therapeutic approach for treatment of cardiometabolic disease. 相似文献
53.
我们采用RT-PCR方法克隆了2个APl同源基因全长cDNA,分别命名为MAPl-1(GenBank accession No.FJ529206)和MAPl-2(GenBank accession No.FJ529207).MAPl-1编码247个氨基酸,开放阅读框长度为741 bp,蛋白质分子量为28.54kD,等电点为8.31;MAPl-2编码248个氨基酸,开放阅读框长度为744 bp,蛋白质分子量为28.78 kD,等电点为8.70.同源性分析表明,它们的核苷酸序列与其它木本植物APl同源基因的一致性为72%~81%.实验分析表明,MAPl-1和MAPl-2第1至第61个氨基酸含有一个MADS盒结构域,第88至第178个为K盒结构域;两个基因均定位于细胞核,且功能位点分布存在着不同,推测这两个基因在花器官发育过程中的功能存在差异.蛋白二级结构预测显示,MAPl-1蛋白有12个a-螺旋,4个β折叠区,14个β-转角;而MAPl-2蛋白有11个a-螺旋,5个β折叠区,15个β-转角:其大多数氨基酸具有亲水性.本研究有助于进一步了解芒果的开花分子机理及成花的生物学发育阶段. 相似文献
54.
含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究 总被引:2,自引:0,他引:2
棕色固氮菌(Azotobacter vinelandii)固氮酶钼铁蛋白经邻菲口罗啉和O2 处理后,变为部分缺失FeMoco 和P-cluster的失活蛋白。与由K2CrO4、高柠檬酸铁、Na2S和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬(CrFe)蛋白 相似文献
55.
松口蘑菌丝体的分离和RAPD-PCR分析 总被引:27,自引:0,他引:27
针对松口蘑 [Tricholomamatsutake(S .ItoetImai)Sing .]菌丝体分离培养困难和各种相关分离物目前难以用出菇试验鉴定的现实 ,采用 8种培养基配方 ,对 9个不同来源的松口蘑子实体的不同部位及菌根、菌土进行组织分离 ,计接种试管 81 0多支 ,结果从菌褶部位获得 94支慢生型的菌丝体分离菌株 ,从菌柄部位仅获得 1支快生型的菌丝体分离菌株。以马铃薯葡萄糖土壤滤液培养基 (PDAS)、马铃薯葡萄糖麦麸滤液培养基 (PDAW )、BM培养基、马铃薯葡萄糖琼脂培养基 (PDA)对菌褶进行组织分离 ,获慢生型菌丝体的成功率依次为 74.4%、35.5%、156%和 8.9%。以各分离菌株的来源松口蘑子实体和中日两国松口蘑研究者提供的分离菌株作为DNA参照样品 ,对从供试子实体、菌根、菌土进行组织分离获得的各种相关纯培养物进行亲菌鉴定。采用筛选的 1 7个随机引物介导 2 5个供试松口蘑子实体及其分离菌体的RAPD(RandomAmplifiedPolymorphicDNA) PCR反应 ,全部获得了清晰而稳定的DNA指纹图谱 ,结果一致表明 :每个松口蘑子实体的菌盖 (含菌褶… 相似文献
56.
Zhong L Guo XN Zhang XH Wu ZX Luo XM Jiang HL Lin LP Zhang XW Ding J 《Biochimica et biophysica acta》2005,1722(3):254-261
Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor. 相似文献
57.
Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg2?, Fe3?, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5-10% inhibition) were observed in the presence of Mn2?, Zn2?, Cu2?, Mg2?, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min?1, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus. 相似文献
58.
Xuelong Lu Yang Shi Quanlong Lu Yan Ma Jia Luo Qingsong Wang Jianguo Ji Qing Jiang Chuanmao Zhang 《The Journal of biological chemistry》2010,285(43):33281-33293
Lamin B receptor (LBR), a chromatin and lamin B-binding protein in the inner nuclear membrane, has been proposed to target the membrane precursor vesicles to chromatin mediated by importin β during the nuclear envelope (NE) assembly. However, the mechanisms for the binding of LBR with importin β and the membrane targeting by LBR in NE assembly remain largely unknown. In this report, we show that the amino acids (aa) 69–90 of LBR sequences are required to bind with importin β at aa 45–462, and the binding is essential for the NE membrane precursor vesicle targeting to the chromatin during the NE assembly at the end of mitosis. We also show that this binding is cell cycle-regulated and dependent on the phosphorylation of LBR Ser-71 by p34cdc2 kinase. RNAi knockdown of LBR causes the NE assembly failure and abnormal chromatin decondensation of the daughter cell nuclei, leading to the daughter cell death at early G1 phase by apoptosis. Perturbation of the interaction of LBR with importin β by deleting the LBR N-terminal spanning region or aa 69–73 also induces the NE assembly failure, the abnormal chromatin decondensation, and the daughter cell death. The first transmembrane domain of LBR promotes the NE production and expansion, because overexpressing this domain is sufficient to induce membrane overproduction of the NE. Thus, these results demonstrate that LBR targets the membrane precursor vesicles to chromatin by interacting with importin β in a LBR phosphorylation-dependent manner during the NE assembly at the end of mitosis and that the first transmembrane domain of LBR promotes the LBR-bearing membrane production and the NE expansion in interphase. 相似文献
59.
60.
Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50% ammonium sulfate fractions,ion exchange chromatography Of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.The purification fold was 464 times and specified activity was 3936 U/mg.Optimum pH value of the phosphatase was 10.5,and was stable between pH 7.5 and 11.The optimum temperature of the phosphatase was 40℃ and it was unstable over 50℃.Km value of the phosphatase was 1.25 mmol/L.In a given condition,the phosphatase was selectively modified by PCMB,NBS,PMSE TNBS,SUAN,DTT,BrAc,and IAc,the results indicate that PMSF,SUA,BrAc,IAc,and TNBS could Obviously inhibit the activity of the phosphatase,and the degree of inhibition depended on the concentration of these reagents.There was little effect on the activity of phosphatase after treatment by PMSF,DTT,and NBT.We primarily conclude that mercapto and imidazole are essential for AKP from silkworm.Also,Lys residue and disulfide bands are necessary to protect the catalysis of the AKP. 相似文献