Separation of 50 S ribosomal proteins from sulfolobus acidocaldarius by discontinuous reversed-phase chromatography

Summary The total protein of the 50S ribosomal subunit (TP50) from the archaebacterium Sulfolobus acidocaldarius was prefractionated by discontinuous reversed-phase HPLC with several column combinations. The purity of the eluted fractions was tested by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE (2D-PAGE). With regard to the load capacity and selectivity, best results were obtained with a semi-preparative three-column combination (total length 67.5cm). Twelve A₂₃₀ units of TP50 were separated into 35 fractions, 19 of which contained nearly pure proteins. When the sample load was increased to 176A₂₃₀ units (109mg), 13 of 28 collected fractions still contained nearly pure proteins. The selectivity of a semi-preparative short-column combination (total length 12cm) was similar to that of the semi-preparative column combination, and separation time could be reduced to one third of that required for the longer column combination. The load capacity of the short-column combination was lower than that of the semi-preparative column combination.     

浙江两地产索氏桃花水母核糖体小亚基rRNA基因序列分析

采用PCR和DNA测序技术,对杭州与绍兴产桃花水母标本的核糖体小亚基rRNA基因进行了扩增与测序．经序列比对分析,杭州与绍兴产桃花水母的核糖体小亚基rRNA基因序列与已知索氏桃花水母的序列极为相似,相似度分别为99．61％和99．45％;与索氏桃花水母的遗传距离均为0．001;经分子系统树分析,杭州与绍兴产桃花水母与索氏桃花水母的分支置信度高达1000．4．综上分析,杭州与绍兴产桃花水母均为索氏桃花水母（Craspedacusta sowerbyi）,该结论与其形态学分类相一致．     

Hypoxia induces Wee1 expression and attenuates hydrogen peroxide-induced endothelial damage in MS1 cells

In an oxygen-depleted environment, endothelial cells initiate an adaptive pattern of synthesis, which may enable them to survive hypoxic crises. Using high-resolution two-dimensional gel electrophoresis in conjunction with mass spectroscopy, we obtained a 24 differential display of proteins in the pancreatic endothelial cell line, MS-1, at four time points following induction of hypoxia. The induction of Wee1 under hypoxia was confirmed both at the mRNA and protein levels. The phosphorylation of cell division cycle 2, which is downstream of Wee1, was also increased after hypoxic exposure. In addition, pre-exposure to hypoxia attenuated a decrease in hydrogen peroxide-induced cell number. The induction of bax (a pro-apoptotic protein) and reduction of bcl (an anti-apoptotic protein) after hypoxia stimulus were also attenuated by hypoxic pre-exposure. Moreover, hydrogen peroxide-induced morphologic damage did not appear in the wild-type Wee1-expressing cells. Taken together, our results suggest that Wee1 may have important role in hypoxia- induced pathophysiological situations in endothelial cells.     

Comparison between the behavior of different hydrophobic peptides allowing membrane anchoring of proteins

Membrane binding of proteins such as short chain dehydrogenase reductases or tail-anchored proteins relies on their N- and/or C-terminal hydrophobic transmembrane segment. In this review, we propose guidelines to characterize such hydrophobic peptide segments using spectroscopic and biophysical measurements. The secondary structure content of the C-terminal peptides of retinol dehydrogenase 8, RGS9-1 anchor protein, lecithin retinol acyl transferase, and of the N-terminal peptide of retinol dehydrogenase 11 has been deduced by prediction tools from their primary sequence as well as by using infrared or circular dichroism analyses. Depending on the solvent and the solubilization method, significant structural differences were observed, often involving α-helices. The helical structure of these peptides was found to be consistent with their presumed membrane binding. Langmuir monolayers have been used as membrane models to study lipid–peptide interactions. The values of maximum insertion pressure obtained for all peptides using a monolayer of 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE) are larger than the estimated lateral pressure of membranes, thus suggesting that they bind membranes. Polarization modulation infrared reflection absorption spectroscopy has been used to determine the structure and orientation of these peptides in the absence and in the presence of a DOPE monolayer. This lipid induced an increase or a decrease in the organization of the peptide secondary structure. Further measurements are necessary using other lipids to better understand the membrane interactions of these peptides.     

Probing the Subunit-Subunit Interaction of the Tetramer of E. coli KDO8P Synthase by Electrospray Ionization Mass Spectrometry

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation reaction between D-arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (Pi). This enzyme exists as a tetramer in solution, which is important for catalysis. Two different states of the enzyme were obtained: i) PEP-bound and ii) PEP-unbound. The effect of the substrates and products on the overall structure of KDO8P synthase in both PEP-bound and unbound states was examined using electrospray ionization mass spectrometry. The analysis of our data showed that the complexes of the PEP-unbound enzyme with PEP (or Pi) favored the formation of monomers, while the complexes with A5P (or KDO8P) mainly favored dimers. The PEP-bound enzyme was found to exist in the monomer and dimer with a small amount of the tetramer, whereas the PEP-unbound form primarily exists in the monomer and dimer, and no tetramer was observed, suggesting that the bound PEP have a role in stabilization of the tetrameric structure. Taken together, the results imply that the addition of the substrates or products to the unbound enzyme may alter the subunit-subunit interactions and/or conformational change of the protein at the active site, and this study also demonstrates that the electrospray ionization mass spectrometric method may be a powerful tool in probing the subunit-subunit interactions and/or conformational change of multi-subunit protein upon binding to ligand.