拥挤环境对碱性磷酸酶去折叠过程的影响作用研究

为了更好地模拟细胞内的真实环境,在碱性磷酸酶的变性环境中分别加入小分子渗透剂（蔗糖）和大分子拥挤试剂（葡聚糖）,创造拥挤环境,利用酶活力测定和荧光光谱等方法,研究该拥挤环境对碱性磷酸酶稳定性的影响作用．实验结果表明:在碱性磷酸酶由盐酸胍导致的变性过程中,蔗糖与葡聚糖可抑制碱性磷酸酶活力的丧失,随着蔗糖与葡聚糖的质量浓度增加,其抑制效果随之增强;荧光光谱的检测结果也表明蔗糖和葡聚糖对碱性磷酸酶的去折叠具有一定的抑制作用．在2种试剂中,蔗糖对碱性磷酸酶活力与构象稳定性的保护能力要强于葡聚糖．      

重金属 Cu 对土壤碱性磷酸酶活性的影响?

采用室内恒温恒湿培养方法,研究不同深度(表层、50、100cm)土壤样品中重金属Cu对碱性磷酸酶(ALP)活性及其动力学特征的影响.结果表明:ALP活性随着培养时间的延长而增加;相同培养时间条件下,随着Cu质量分数w的增加,表层、50cm样品的土壤ALP活性呈现先升高后降低的变化趋势,100cm样品的土壤ALP活性减小;不同土壤ALP活性变化存在差异,这与各土壤样品的理化性质,尤其是有机质含量不同有关;w(Cu)与ALP的Vmax、Vmax/Km显著相关,可以考虑将ALP催化动力学参数Vmax、Vmax/Km值作为辅助参考指标用作考察土壤重金属Cu污染水平;金属Cu对ALP的作用先是非竞争性抑制,后是竞争性抑制,表现出一种混合抑制类型.     

Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants

The potential impact of pandemic influenza makes effective measures to limit the spread and morbidity of virus infection a public health priority. Antiviral drugs are seen as essential requirements for control of initial influenza outbreaks caused by a new virus, and in pre-pandemic plans there is a heavy reliance on drug stockpiles. The principal target for these drugs is a virus surface glycoprotein, neuraminidase, which facilitates the release of nascent virus and thus the spread of infection. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two currently used neuraminidase inhibitors that were developed using knowledge of the enzyme structure. It has been proposed that the closer such inhibitors resemble the natural substrate, the less likely they are to select drug-resistant mutant viruses that retain viability. However, there have been reports of drug-resistant mutant selection in vitro and from infected humans. We report here the enzymatic properties and crystal structures of neuraminidase mutants from H5N1-infected patients that explain the molecular basis of resistance. Our results show that these mutants are resistant to oseltamivir but still strongly inhibited by zanamivir owing to an altered hydrophobic pocket in the active site of the enzyme required for oseltamivir binding. Together with recent reports of the viability and pathogenesis of H5N1 (ref. 7) and H1N1 (ref. 8) viruses with neuraminidases carrying these mutations, our results indicate that it would be prudent for pandemic stockpiles of oseltamivir to be augmented by additional antiviral drugs, including zanamivir.     

Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide

Proteins secreted by prokaryotic cells are synthesized as precursors containing an amino-terminal extension sequence or signal peptide. Although these signal peptides share little primary sequence homology, recent studies suggest that they function via common pathways during the transport process and that a common element may reside in their secondary structural characteristics. We are investigating the role of an idealized hydrophobic sequence with high potential for alpha-helix formation in the Escherichia coli alkaline phosphatase signal peptide. Here, amino-acid substitutions were made using site-directed mutagenesis to produce a mutant signal sequence containing nine consecutive leucine residues in the hydrophobic core segment. Transport studies with this mutant precursor indicate that mature alkaline phosphatase is correctly targeted to the E. coli periplasm and that processing of the precursor to the mature form of the enzyme is extremely rapid. In contrast, processing is slowed when the mutant signal sequence is lengthened by the insertion of five additional leucine residues and one serine.