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91.
Lisa Klug Pablo Tarazona Clemens Gruber Karlheinz Grillitsch Brigitte Gasser Martin Trötzmüller Harald Köfeler Erich Leitner Ivo Feussner Diethard Mattanovich Friedrich Altmann Günther Daum 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2014,1841(2):215-226
The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications. 相似文献
92.
93.
Madeleine Strickland Oscar Juárez Yashvin Neehaul Darcie A. Cook Blanca Barquera Petra Hellwig 《The Journal of biological chemistry》2014,289(34):23723-23733
Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na+-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na+-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active. 相似文献
94.
Silke Haerteis Annabel Krappitz Matteus Krappitz Jane E. Murphy Marko Bertog Bettina Krueger Regina Nacken Hyunjae Chung Morley D. Hollenberg Wolfgang Knecht Nigel W. Bunnett Christoph Korbmacher 《The Journal of biological chemistry》2014,289(27):19067-19078
Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases. 相似文献
95.
Octavian C?linescu Cristina Paulino Werner Kühlbrandt Klaus Fendler 《The Journal of biological chemistry》2014,289(19):13168-13176
Na+/H+ exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na+/H+ exchangers, where a single binding site is alternatively occupied by Na+ or one or two H+ ions. The proposed transport mechanism inherently down-regulates Na+/H+ exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na+/H+ exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na+/H+ exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na+/H+ exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na+/H+ exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na+/H+ exchangers. 相似文献
96.
Jacobo Elies Mark L. Dallas John P. Boyle Jason L. Scragg Adrian Duke Derek S. Steele Chris Peers 《The Journal of biological chemistry》2014,289(23):16421-16429
Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, l-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor Nω-nitro-l-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state. 相似文献
97.
Qian Liu Yao JinKun Wang Xiang-Xue MengYang Yang Zhuo YangYong-Shan Zhao Ming-Yi ZhaoJing-Hai Zhang 《Comptes rendus biologies》2014,337(2):73-77
The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role. 相似文献
98.
异常表达的糖蛋白与PD等多种神经退行性疾病有关。糖蛋白组学研究发现,电压门控钠离子通道β4亚基在PD病人脑组织中表达明显增加。为了深入探索β4亚基及其糖链在帕金森发生发展中的作用,采用PD转基因鼠对其表达进行验证,对其潜在的糖基化位点进行定点突变,构建重组表达质粒。结果发现,在新生PD转基因鼠和野生成鼠脑组织中有~38kDa蛋白条带表达,而在新生野生鼠脑组织中不表达;用PNGase F酶处理去除糖链后,~38kDa蛋白条带变成迁移速递更快的较小分子量条带,说明β4亚基是高度糖基化的蛋白,并且其糖基化与生长发育有关。将突变重组质粒转入HEK-293细胞和小鼠神经瘤细胞Neuro2A中表达,结果发现突变型质粒分子量明显低于野生型。为研究β4亚基及其糖链的功能提供了一定的实验数据并打下了基础。 相似文献
99.
A. G. Maaurf Y. B. Che Man B. A. Asbi A. H. Junainah J. F. Kennedy 《Carbohydrate polymers》2001,45(4):381-345
The inhibitory effect of sucrose and sodium chloride on sago starch gelatinisation was investigated by differential scanning calorimetry (DSC). The temperature of gelatinisation of starch in the presence of low levels of water and high levels of sucrose was found to increase in the presence of sucrose, whereas the gelatinisation enthalpy was unaffected. The gelatinisation temperature range was not as broad in the presence of sucrose as without sucrose. Furthermore, the shape of the gelatinisation endotherm was changed by the addition of sucrose. The double endotherm obtained in limited water:starch systems was changed into a single endotherm, similar to the endotherm obtained in excess water:starch systems at a higher temperature. DSC was also used to examine the effects of water and sodium chloride content on the phase transitions of sago starch. Samples were adjusted to starch:water ratios of 2:3 and 3:2 in sodium chloride concentrations of 0.0, 1.0, 2.0, 3.0, 4.0, 5.0 M. The gelatinisation temperatures of sago starch increased and then decreased as the sodium chloride concentration increased. Sodium chloride created similar effects on the endotherms in excess water content and on the first endotherm with limited water content. In the presence of sucrose and sodium chloride, gelatinisation shifted to higher temperatures, and enthalpy associated with the endothermic process decreased. The extent of temperature shift and enthalpy change was dependent on the water to starch to solutes ratios. 相似文献
100.
Banghua Sun Peizhang Xu Weiya Wang Paul M. Salvaterra 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,130(4)
We have constructed and characterized transgenic Drosophila lines with modified Na+,K+-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na+,K+-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (αD369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system we have limited expression of α transgenes to cell types that normally express a particular Drosophila Na+,K+-ATPase β (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types. 相似文献