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991.
992.
Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site-specific protein phosphorylation degree determination by nanoLC-ESI-MS/MS. A stable isotope-labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one-source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one-source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6. 相似文献
993.
Mara L. Leimanis Joel Karwatsky Elias Georges 《International Journal of Biochemistry and Molecular Biology》2011,2(1):39-46
Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC4); however direct binding of the LTC4 to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC4 (IAALTC4) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC4 in plasma membranes from MDCKIIMRP2 cells. The photoaffinity labeling signal of MRP2 with IAALTC4 was much lower than that of MRP1, consistent with previous studies whereby the measured Km values of MRP1 and MRP2 for LTC4 were 1 μM and 0.1 μM LTC4, respectively. Competition of IAALTC4 photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 μM excess MK571. Interestingly, unmodified LTC4 enhanced the photoaffinity labeling of IAALTC4 to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC4 binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC4 binding to MRP2. 相似文献
994.
995.
Paul L. Prather FeAna FrancisDevaraj Centdrika R. Dates Aleksandra K. Greer Stacie M. Bratton Benjamin M. Ford Lirit N. Franks Anna Radominska-Pandya 《Biochemical and biophysical research communications》2013
Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9–3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs. 相似文献
996.
Olaf Maier Roman Fischer Cristina Agresti Klaus Pfizenmaier 《Biochemical and biophysical research communications》2013
The neuroprotective role of TNF receptor 2 (TNFR2) has been shown in various studies. However, a direct role of TNFR2 in oligodendrocyte function has not yet been demonstrated. Using primary oligodendrocytes of transgenic mice expressing human TNFR2, we show here that TNFR2 is primarily expressed on oligodendrocyte progenitor cells. Interestingly, preconditioning with a TNFR2 agonist protects these cells from oxidative stress, presumably by increasing the gene expression of distinct anti-apoptotic and detoxifying proteins, thereby providing a potential mechanism for the neuroprotective role of TNFR2 in oligodendrocyte progenitor cells. 相似文献
997.
998.
Jonathan P. Badalamenti César I. Torres Rosa Krajmalnik‐Brown 《Biotechnology and bioengineering》2013,110(4):1020-1027
The objective of this study was to employ microbial electrochemical cells (MXCs) to selectively enrich and examine anoxygenic photosynthetic bacteria for potential anaerobic respiration capabilities using electrodes. In the process, we designed a novel enrichment strategy that manipulated the poised anode potential, light, nitrogen availability, and media supply to promote growth of phototrophic bacteria while minimizing co‐enrichment of non‐phototrophic anode‐respiring bacteria (ARB). This approach resulted in light‐responsive electricity generation from fresh‐ and saltwater inocula. Under anoxic conditions, current showed a negative light response, suggesting that the enriched phototrophic consortia shifted between phototrophic and anaerobic respiratory metabolism. Molecular, physical, and electrochemical analyses elucidated that anode biofilms were dominated by green sulfur bacteria, and biofilms exhibited anode respiration kinetics indicative of non‐mediated electron transfer, but kinetic parameters differed from values previously reported for non‐phototrophic ARB. These results invite the utilization of MXCs as microbiological tools for exploring anaerobic respiratory capabilities among anoxygenic photosynthetic bacteria. Biotechnol. Bioeng. 2013; 110: 1020–1027. © 2012 Wiley Periodicals, Inc. 相似文献
999.
《Peptides》2013
Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe120 in the first extracellular loop. This was in contrast to its covalent attachment to Glu345 in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors. 相似文献
1000.
Mao-Yu Li Fang Peng Jian-Hong Zuo Hong Yi Can-E Tang Cui Li Peng-Fei Zhang Zhu-Chu Chen Zhi-Qiang Xiao 《Analytical biochemistry》2011,(1):37
Trypsin-catalyzed 18O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of 18O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of 18O-labeled peptides under various pH conditions. The 18O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient–isoelectric focusing (IPG–IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of 18O labeling and molecular weight or isoelectric point (pI). The approach presented here is especially applicable to microscale samples. Its ability to generate stably 18O-labeled samples without back-exchange should expand the application scope of the 18O-labeling technique. 相似文献