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排序方式: 共有217条查询结果,搜索用时 15 毫秒
91.
Seibert C Veldkamp CT Peterson FC Chait BT Volkman BF Sakmar TP 《Biochemistry》2008,47(43):11251-11262
CXC-chemokine receptor 4 (CXCR4) is a G protein-coupled receptor for stromal cell-derived factor-1 (SDF-1/CXCL12). SDF-1-induced CXCR4 signaling is indispensable for embryonic development and crucial for immune cell homing and has been implicated in metastasis of numerous types of cancer. CXCR4 also serves as the major coreceptor for cellular entry of T-cell line-tropic (X4) HIV-1 strains. Tyrosine residues in the N-terminal tail of CXCR4, which are post-translationally sulfated, are implicated in the high-affinity binding of SDF-1 to CXCR4. However, the specific roles of three potential tyrosine sulfation sites are not well understood. We investigated the pattern and sequence of CXCR4 sulfation by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a peptide that corresponds to amino acids 1-38 of the receptor (CXCR4 1-38). We analyzed the reaction products with a combination of reversed-phase HPLC, proteolytic cleavage, and mass spectrometry. We found that CXCR4 1-38 is sulfated efficiently by both TPST enzymes, leading to a final product with three sulfotyrosine residues. Sulfates were added stepwise to the peptide, producing specific intermediates with one or two sulfotyrosines. The pattern of sulfation in these intermediates indicates that with both enzymes Tyr-21 is sulfated first, followed by Tyr-12 or Tyr-7. Using heteronuclear NMR spectroscopy, we demonstrated that the SDF-1 binding affinity of CXCR4 1-38 increases with the number of sulfotyrosines present, which suggests a potential physiological role for sulfation of all three sites in the N-terminus of CXCR4. These results provide a structural basis for understanding the role of post-translational tyrosine sulfation in SDF-1-induced CXCR4 signaling. 相似文献
92.
Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances 总被引:30,自引:2,他引:28
Hu Q Sommerfeld M Jarvis E Ghirardi M Posewitz M Seibert M Darzins A 《The Plant journal : for cell and molecular biology》2008,54(4):621-639
Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20–50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization. 相似文献
93.
The differential sensitivity of human and rhesus macaque CCR5 to small-molecule inhibitors of human immunodeficiency virus type 1 entry is explained by a single amino acid difference and suggests a mechanism of action for these inhibitors 总被引:2,自引:0,他引:2
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Billick E Seibert C Pugach P Ketas T Trkola A Endres MJ Murgolo NJ Coates E Reyes GR Baroudy BM Sakmar TP Moore JP Kuhmann SE 《Journal of virology》2004,78(8):4134-4144
AD101 and SCH-C are two chemically related small molecules that inhibit the entry of human immunodeficiency virus type 1 (HIV-1) via human CCR5. AD101 also inhibits HIV-1 entry via rhesus macaque CCR5, but SCH-C does not. Among the eight residues that differ between the human and macaque versions of the coreceptor, only one, methionine-198, accounts for the insensitivity of macaque CCR5 to inhibition by SCH-C. Thus, the macaque coreceptor engineered to contain the natural human CCR5 residue (isoleucine) at position 198 is sensitive to HIV-1 entry inhibition by SCH-C, whereas a human CCR5 mutant containing the corresponding macaque residue (methionine) is resistant. Position 198 is in CCR5 transmembrane (TM) helix 5 and is not located within the previously defined binding site for AD101 and SCH-C, which involves residues in TM helices 1, 2, 3, and 7. SCH-C binds to human CCR5 whether residue 198 is isoleucine or methionine, and it also binds to macaque CCR5. However, the binding of a conformation-dependent monoclonal antibody to human CCR5 is inhibited by SCH-C only when residue 198 is isoleucine. These observations, taken together, suggest that the antiviral effects of SCH-C and AD101 involve stabilization, or induction, of a CCR5 conformation that is not compatible with HIV-1 infection. However, SCH-C is unable to exert this effect on CCR5 conformation when residue 198 is methionine. The region of CCR5 near residue 198 has, therefore, an important influence on the conformational state of this receptor. 相似文献
94.
Probing Photosynthesis on a Picosecond Time Scale: Evidence for Photosystem I and Photosystem II Fluorescence in Chloroplasts
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Fluorescent emission kinetics of isolated spinach chloroplasts have been observed at room temperature with an instrument resolution time of 10 ps using a frequency doubled, mode-locked Nd:glass laser and an optical Kerr gate. At 685 nm two maxima are apparent in the time dependency of the fluorescence; the first occurs at 15 ps and the second at 90 ps after the flash. The intervening minimum occurs at about 50 ps. On the basis of theoretical models, lifetimes of the components associated with the two peaks and spectra (in escarole chloroplasts), the fluorescence associated with the first peak is interpreted as originating from Photosystem I (PSI) (risetime ≤10 ps, lifetime ≤10 ps) and the second peak from Photosystem II (PSII) (lifetime, 210 ps in spinach chloroplasts and 320 ps in escarole chloroplasts). The fact that there are two fluorescing components with a quantum yield ratio ≤0.048 explains the previous discrepancy between the quantum yield of fluorescence measured in chloroplasts directly and that calculated from the lifetime of PSII. The 90 ps delay in the peak of PSII fluorescence is probably explained by energy transfer between accessory pigments such as carotenoids and Chl a. Energy spillover between PSI and PSII is not apparent during the time of observation. The results of this work support the view that the transfer of excitation energy to the trap complex in both photosystems occurs by means of a molecular excitation mechanism of intermediate coupling strength. Although triplet states are not of major importance in energy transfer to PSII traps, the possibility that they are involved in PSI photochemistry has not been eliminated. 相似文献
95.
The "high-affinity Mn-binding site" in Mn-depleted photosystem II (PS II) membrane fragments isolated from Scenedesmus obliquus was examined by using the diphenylcarbazide (DPC)/Mn2+ non-competitive inhibition assay [Preston, C., & Seibert, M. (1991) Biochemistry (preceding paper in this issue)]. Different proteases were used to degrade lumenal surface protein segments from these PS II membranes, and a total of four independent high-affinity Mn-binding sites (ligands) were identified. Carboxypeptidase A, subtilisin, and Staphylococcus aureus V8 protease each degrade one of two high-affinity Mn-binding sites sensitive to the histidine chemical modifier diethyl pyrocarbonate (DEPC). However, sequential treatment experiments indicate that subtilisin degrades a DEPC-sensitive Mn-binding site that is different from the one degraded by the other two proteases. Trypsin also was found to degrade one of the DEPC-sensitive Mn-binding sites (that degraded by carboxypeptidase A and V8 protease). In addition, trypsin degrades one of two 1-ethyl-3-[(3-dimethylamino)propyl]carbodiimide (EDC) sensitive Mn-binding sites, but only in the absence of the 30-kDa extrinsic protein. Thus, the 30-kDa extrinsic protein associated with O2 evolution appears to protect the EDC-sensitive binding site from trypsin degradation. No protease has yet been identified that will degrade the trypsin-insensitive EDC-sensitive Mn-binding site. Under the conditions of the assay (high DPC concentration), more than three Mn per reaction center were found bound to the membrane with a KM of about 0.4 microM, as determined by direct metal analysis. This is consistent with the idea that each of the four high-affinity sites binds (or provides a ligand for) one of four Mn.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
96.
Deming Tang Ryszard Jankowiak Michael Seibert Gerald J. Small 《Photosynthesis research》1991,27(1):19-29
Low temperature (4.2 K) absorption and hole burned spectra are reported for a stabilized preparation (no excess detergent) of the photosystem II reaction center complex. The complex was studied in glasses to which detergent had and had not been added. Triton X-100 (but not dodecyl maltoside) detergent was found to significantly affect the absorption and persistent hole spectra and to disrupt energy transfer from the accessory chlorophyll a to the active pheophytin a. However, Triton X-100 does not significantly affect the transient hole spectrum and lifetime (1.9 ps at 4.2 K) of the primary donor state, P680*. Data are presented which indicate that the disruptive effects of Triton X-100 are not due to extraction of pigments from the reaction center, leaving structural perturbations as the most plausible explanation. In the absence of detergent the high resolution persistent hole spectra yield an energy transfer decay time for the accessory Chl a QY-state at 1.6 K of 12 ps, which is about three orders of magnitude longer than the corresponding time for the bacterial RC. In the presence of Triton X-100 the Chl a QY-state decay time is increased by at least a factor of 50.Abbreviations PS I
photosystem I
- PS II
photosystem II
- RC
reaction center
- P680, P870, P960
the primary electron donor absorption bands of photosystem II, Rhodobacter sphaeroides, Rhodopseudomonas viridis
- NPHB
nonphotochemical hole burning
- TX
Triton X-100
- DM
Dodecyl Maltoside
- Chl
chlorophyll
- Pheo
pheophytin
- ZPH
ero phonon hole 相似文献
97.
Post-translational processing and secretory pathway of human atriopeptin in rat pheochromocytoma cells. 总被引:1,自引:0,他引:1
S Shiono N Suganuma M Bo I Boime K Seibert K Nakao M Mukoyama H Imura P Needleman 《Biochemical and biophysical research communications》1991,176(3):1232-1238
Atriopeptin (AP) is expressed in several tissues with each tissue capable of specific differences in processing of the prohormone (pro-AP) to mature low molecular forms of the peptide. Since pro-AP has low biological activity, processing into mature AP is a critical activation event. This observation prompted us to study whether granule storage or regulated secretion of AP is essential for cleavage of mature peptide. We examined the processing of AP in adrenal medulla derived cells, using the rat pheochromocytoma cell line (PC12 cell) stably transfected with a genomic human AP DNA in the presence and absence of nerve growth factor (NGF), and also examined the mechanism of AP secretion and compared the results with those obtained using transfected chinese hamster ovary cells (CHO cells). The amount of prohormone was 5-10 fold higher than that of low molecular form of AP in the transfected PC12 cells. This ratio was essentially unchanged in differentiated PC12 cells after NGF treatment of the cells. Potassium depolarization of the transfected PC12 cells caused a 5-fold increase in AP release into the medium primarily as the intact prohormone. On the other hand, transfected CHO cells only exhibited constitutive AP release which is non-response to depolarization. These results suggest that the AP prohormone is sorted into secretory granules as the prohormone in PC12 cells and undergoes regulated release in response to depolarization indicating granule storage or release is not the critical determinant of AP prohormone cleavage. 相似文献
98.
99.
Smith JJ Alphy S Seibert AL Blumenthal KM 《The Journal of biological chemistry》2005,280(12):11127-11133
It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV. 相似文献
100.
Grimm R Huber R Neumeier T Seidl A Haslbeck M Seibert FS 《Analytical biochemistry》2004,330(1):140-144
The identification and characterization of a protein overexpressed in insoluble inclusion bodies in Escherichia coli are the first crucial and time-limiting steps in recombinant protein expression. Here, a straightforward approach to the analysis of recombinant proteins in inclusion bodies is presented. Inclusion bodies were dissolved in 8M urea and analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry without prior desalting. Mass determination was achieved by direct spotting of the samples onto the MALDI target and serial dilution in the matrix. The masses of four different proteins, expressed in inclusion bodies, were determined with a mass accuracy better than 0.1%. Furthermore, protein modifications, such as N-terminal processing of single amino acids or artificial cyanylation caused by incubation of the inclusion bodies with urea at elevated temperatures, could be detected. Similarly, tryptic digests were directly analyzed in 2M urea to obtain peptide mass fingerprints for identification and more detailed information on the primary protein structure and secondary modifications. Due to the presence of ammonia in the urea-containing buffers, no Na(+) adducts were observed in the peptide mass fingerprint analysis. Taken together, the rapid and robust procedures presented here greatly facilitate the analysis of recombinant proteins. 相似文献