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2.
Chien-Jung Huang Laurent Schild Edward G. Moczydlowski 《The Journal of general physiology》2012,140(4):435-454
Voltage-gated Na+ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca2+ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca2+ or Na+ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca2+ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation. 相似文献
3.
Ronald J.A. Wanders Carlo Van Roermund Cor Lof Alfred J. Meijer 《Analytical biochemistry》1983,129(1):80-87
A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay, production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP+, and glucose-6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present. 相似文献
4.
In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution. 相似文献
5.
Marvin H. Caruthers Lincoln J. Mcbride Laurent P. Bracco John W. Dubendorff 《Nucleosides, nucleotides & nucleic acids》2013,32(1-2):95-105
Abstract An in situ method is described for synthesizing DNA which incorporates a new series of amidine protected deoxy-nucleosides and bis-dialkylaminophosphines as phosphitylating agents. These procedures were used to synthesize d(GGGAATTCCC) which was digested by EcoRI. 相似文献
6.
Laurent Chatel-Chaix Marie-Anne Germain Alena Motorina éric Bonneil Pierre Thibault Martin Baril Daniel Lamarre 《Journal of virology》2013,87(21):11704-11720
Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. 相似文献
7.
A Merlin W Voos A C Maarse M Meijer N Pfanner J Rassow 《The Journal of cell biology》1999,145(5):961-972
Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain. 相似文献
8.
The rate of proteolysis is an important determinant of the intracellular protein content. Part of the degradation of intracellular
proteins occurs in the lysosomes and is mediated by macroautophagy. In liver, macroautophagy is very active and almost completely
accounts for starvation-induced proteolysis. Factors inhibiting this process include amino acids, cell swelling and insulin.
In the mechanisms controlling macroautophagy, protein phosphorylation plays an important role. Activation of a signal transduction
pathway, ultimately leading to phosphorylation of ribosomal protein S6, accompanies inhibition of macroautophagy. Components
of this pathway may include a heterotrimeric Gi3-protein, phosphatidylinositol 3- kinase and p70S6 kinase. Recent evidence
indicates that lysosomal protein degradation can be selective and occurs via ubiquitin- dependent and -independent pathways.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
9.
10.
Regulation of autotrophic metabolism in Pseudomonas oxalaticus OX1 wild-type and an isocitrate-lyase-deficient mutant 总被引:1,自引:0,他引:1
In Pseudomonas oxalaticus the activity and synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) are regulated by inactivation and endproduct repression, respectively. Phosphoenolpyruvate (PEP) has been suggested to function as a signal molecule for the latter control system. During growth of the organism in carbon-source-limited continuous cultures with various ratios of acetate and formate in the feed, the RuBisCO levels varied considerably, but no correlation was observed with the intracellular concentrations of PEP. To study whether the repression exerted by acetate utilization was dependent on the synthesis of glycolytic intermediates from this compound, an acetate-negative mutant defective in isocitrate lyase was isolated and characterized. Clear evidence was obtained that in this mutant acetate is as effective in repressing RuBisCO synthesis as in the wild-type. It therefore appears more likely that acetyl-CoA or a closely related metabolite functions as a signal molecule in the regulation of RuBisCO synthesis. 相似文献