Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与HSA相互作用的~(23)(Na)-NMR研究

 本文应用~23Na-NMR波谱技术,研究了Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与人体血清白蛋白(HSA)的相互作用。在实验基础上,通过引入两位快交换模型,拟合计算获得了Na~(+)与HSA相互作用的结合常数和处于结合状态Na~(+)的相关时间;实验表明Ca~(2+)能与Na~(+)竞争同HSA结合,拟合计算获得了两者与HSA相互作用结合常数的比值,棕榈酸钠能增强Ca~(2+)同Na~(+)竞争与HSA结合的能力;从实验上未能观察到Cu~(2+)、Zn~(2+)能同Na~(+)竞争与HSA相互作用的证据。     

双胸蚓纤溶酶的纯化及性质

 用硫酸铵分段盐析、超滤膜分级分离及DEAE-纤维素、Sephadex A-25和Sephadex G-50三种柱层析方法从双胸蚓组织的粗提取液中分离纯化出一种纤溶酶,分子量为29kD,由一条肽链组成。此晦具有强烈的溶解纤维蛋白的作用,对家兎实验性血凝块也具有明显的溶解作用。此酶的最适pH为8.0,在pH7.6～8.4之间活力相差不到2％;酶在PH4.7—11.0范围内稳定;酶作用的最适温度为57℃;此酶热稳定性较好,于25～50℃保温3小时,酶活力基本不变,60℃时,活力保留65％。金属离子Na~(+)、K~(+)、Mg~(2+)等可提高此酶的活力,而Hg~(2+)、Ca~(2+)等金属离子对此酶有不同程度的抑制作用。     

化学法合成生物素标记寡核苷酸探针

 生物素可通过氨烷基磷酰胺基团连接到寡核苷酸5'末端,反应在水溶液中进行,核苷酸的侧链基团不用保护。我们以这种化学法合成了生物素标记的寡核苷酸探针,其显色灵敏度达50pg,杂交灵敏度达0.4fmol,并与酶标生物素寡核苷酸探针进行了比较,也对两种不同显色体系作了比较。     

外源脂肪酸及胆固醇对莱氏衣原体膜糖脂含量的影响

 <正> 单葡萄糖甘油二酯(MGDG)和双葡萄糖甘油二酯(DGDG)是莱氏衣原体膜上主要的极性脂。糖脂的生理功能过去很少研究,至今仍不清楚。近年来实验结果表明,MGDG在膜上容易形成六角形Ⅱ结构,而DGDG则形成脂双层结构。六角形Ⅱ结构的出现与生物膜的生理功能的关系是当前瞩目的研究内容。本文首次研究了外源脂肪酸和胆固酵对莱氏衣原体AIH089菌株膜上糖脂含量的影响。     

不同底物与固氮酶及其钼铁蛋白相互作用的荧光光谱分析

 本文研究了不同底物(N_2,H_2,N_2O,NaN_3,C_2H_2)对棕色固氮菌固氮酶及其钼铁蛋白荧光光谱的影响。结果表明,上述底物均能络合在钼铁蛋白及固氮酶上,但络合程度不同,从而为固氮酶系统有多个不同的底物络合中心,底物络合中心在钼铁蛋白分子上,铁蛋白对钼铁蛋白有变构作用,提供了光谱学证据。     

固定化谷氨酸棒杆菌T6—13细胞的酶学性质研究

比较研究了固定化谷氨酸棒杆菌细胞和自然细胞的谷氨酸脱氢酶、异拧檬酸脱氢酶,葡萄糖-6-磷酸脱氢酶的一些性质。最适pH、温度对二者酶促反应速度的影响基本相似;pH、热稳定性固定化细胞高于自然细胞;底物表观米氏常数谷氨酸脱氢酶,异柠檬酸脱氢酶有所增大,而葡萄糖-6-磷酸脱氢酶则有所下降;辅酶表观米氏常数均有所增大。这些是影响固定化细胞应用的主要因素。     

Regulation of intracellular calcium in epithelial cells

The role of [Ca2+]i as a second messenger in non-excitable cells has been appreciated for almost 3 decades. The advent of fluorescent Ca2+ indicators has allowed the monitoring of Ca2+ signalling in suspensions of these cells. Agonist mediated changes in [Ca2+]i usually show an initial Ca2+ transient followed by a maintained increase. The former has been shown to be due to Ca2+ release from one or more intracellular stores, the latter due to activation of receptor operated Ca2+ entry (ROCE). More recently it has been recognized that many cells show distinct maintained oscillatory behavior when examined by single cell optical methods. It is proposed here that these oscillations are the consequence of IP3 and Ca2+ stimulation of Ca2+ release and ligand activation of ROCE followed by Ca2+ inhibition of Ca2+ and ROCE as Ca2+ pumps are activated. These oscillations allow more exact regulation of a pump/leak controlled second messenger such as [Ca2+]i.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies.

Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ration in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means +/- standard errors) 41 +/- 10 and 37 +/- 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.