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ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release.
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We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell. 相似文献
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Klaus-Günther Barthel 《Polar Biology》1990,10(5):343-350
Summary During MIZEX'83 and MIZEX'84 food composition and food uptake by three Calanus species were investigated in the northern Greenland Sea. Samples were obtained from open water, the marginal ice zone, the pack ice region and the East Greenland shelf polynya. Food uptake of Calanus spp. was determined in ship-board incubation experiments under in situ conditions and volume of particulate matter in the upper 300 m of the water column was measured. Principal components analysis was used for data evaluation. Under the pack ice strongly reduced amounts of particulate matter in the euphotic zone were found while concentrations were elevated in the marginal ice zone and the East Greenland shelf polynya. Food uptake of C. finmarchicus was correlated to food concentration. In the pack ice region ingestion was close to zero for all species investigated. Likewise body weights were significantly lower than in MIZ or polynya. Principal components analysis of physical data shows a clustering of MIZ station groups at high temperature and salinity, clearly separated from pack ice and polynya stations. Analysis of biological data results in the same grouping of stations demonstrating a strong influence of hydrographic conditions on plankton development. It is concluded that herbivorous copepods cannot sustain themselves in the pack ice region but only at places of high productivity like marginal ice zone and East Greenland shelf polynya. 相似文献
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We have analyzed a total of 12 different global and local multiple
protein-sequence alignment methods. The purpose of this study is to
evaluate each method's ability to correctly identify the ordered series of
motifs found among all members of a given protein family. Four
phylogenetically distributed sets of sequences from the hemoglobin, kinase,
aspartic acid protease, and ribonuclease H protein families were used to
test the methods. The performance of all 12 methods was affected by (1) the
number of sequences in the test sets, (2) the degree of similarity among
the sequences, and (3) the number of indels required to produce a multiple
alignment. Global methods generally performed better than local methods in
the detection of motif patterns.
相似文献
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A rapid two-step procedure has been developed for the purification of Despro(2)-Val15-Leu17-aprotinin from the culture supernatant of a recombinant yeast by affinity and ion-exchange chromatography. DesPro(2)-Val15-Leu17-aprotinin was purified to homogeneity, as demonstrated by dodecylsulfate gel electrophoresis and analysis of the N-terminal amino acid sequence. (c) 1993 John Wiley & Sons, Inc. 相似文献
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Marijn Bauters Sofie Meeus Matti Barthel Piet Stoffelen Hannes P. T. De Deurwaerder Flicien Meunier Travis W. Drake Quentin Ponette Jerme Ebuy Pieter Vermeir Hans Beeckman Francis wyffels Samuel Bod Hans Verbeeck Filip Vandelook Pascal Boeckx 《Global Change Biology》2020,26(8):4449-4461
Forests exhibit leaf‐ and ecosystem‐level responses to environmental changes. Specifically, rising carbon dioxide (CO2) levels over the past century are expected to have increased the intrinsic water‐use efficiency (iWUE) of tropical trees while the ecosystem is gradually pushed into progressive nutrient limitation. Due to the long‐term character of these changes, however, observational datasets to validate both paradigms are limited in space and time. In this study, we used a unique herbarium record to go back nearly a century and show that despite the rise in CO2 concentrations, iWUE has decreased in central African tropical trees in the Congo Basin. Although we find evidence that points to leaf‐level adaptation to increasing CO2—that is, increasing photosynthesis‐related nutrients and decreasing maximum stomatal conductance, a decrease in leaf δ13C clearly indicates a decreasing iWUE over time. Additionally, the stoichiometric carbon to nitrogen and nitrogen to phosphorus ratios in the leaves show no sign of progressive nutrient limitation as they have remained constant since 1938, which suggests that nutrients have not increasingly limited productivity in this biome. Altogether, the data suggest that other environmental factors, such as increasing temperature, might have negatively affected net photosynthesis and consequently downregulated the iWUE. Results from this study reveal that the second largest tropical forest on Earth has responded differently to recent environmental changes than expected, highlighting the need for further on‐ground monitoring in the Congo Basin. 相似文献
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The African Black Oystercatcher Haematopus moquini is a charismatic, southern African near-endemic, wader species, that is often seen as a flagship species for coastal bird conservation, as it was recently down-listed regionally to Least Concern on the IUCN Red List of Threatened Species. To celebrate this rare conservation success story, BirdLife South Africa named it the 2018 Bird of the Year and ran a year-long programme in collaboration with the Nature’s Valley Trust highlighting aspects of the species’ biology, current threats, and conservation success. We used data collected by the Southern African Bird Atlas Project (SABAP1 and SABAP2) to examine changes in the species’ range and relative abundance, both in the records between the two projects, as well as trends within the SABAP2 sampling period (2008–2017). This case study enabled us to assess whether such metrics can accurately reflect abundance and distributional changes in a species. We found increases in the reported range and the reporting rates between the two Atlas projects, and that the SABAP2 reporting rate was stable. Regionally, across four coastal categories, the reporting rate was lowest in KwaZulu-Natal, though this region also showed an increase in the probability of reporting during the SABAP2 period. While corroborating the recent change in the species’ conservation status, we also provide good evidence that the long-term SABAP data can be used successfully to assess population trends and range changes over time. 相似文献
10.
Preparations of Escherichia coli DnaK from our lab as well as preparations of DnaK and other HSP70 proteins from several major labs in the field produce a stoichiometric initial burst of [alpha-(32)P]ADP when incubated with [alpha-(32)P]ATP and contain an ADP kinase activity. We determined that the initial burst activity results from the transfer of gamma-phosphate from the radiolabeled substrate [alpha-(32)P]ATP to unlabeled ADP bound by the DnaK and is the same activity that results in ADP phosphorylation. The purification of DnaK from E. coli cells that carry a disrupted ndk gene, ndk::km, results in preparations with greatly reduced ADP kinase activities compared with preparations of DnaK purified from ndk(+) cells. The reduction in the amount of ADP kinase activity in preparations of DnaK purified from ndk::km cells shows that nucleoside-diphosphate kinase (NDP kinase) is responsible for most of the ADP kinase activity present in DnaK preparations isolated from ndk(+) cells. The remaining ADP kinase activity in preparations from ndk::km cells, which varies between preparations, is also a property of NDP kinase, which is most likely expressed because of a low frequency reversion of the disrupted ndk gene. A weak, but measurable physical interaction exists between DnaK and NDP kinase and may be at least partially responsible for the co-purification of NDP kinase with DnaK. The presence of contaminating NDP kinase can explain the range of k(cat) values reported for the ATPase activity of DnaK as well as recent reports of initial burst kinetics by DnaK (Banecki, B., and Zylicz, M. (1996) J. Biol. Chem. 271, 6137-6143) and an ADP-ATP exchange activity of DnaK (Hiromura, M., Yano, M., Mori, H., Inoue, M., and Kido, H. (1998) J. Biol. Chem. 273, 5435-5438). 相似文献