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The stimulation of [3H]glucose release (a measure of glycogenolysis) from isolated guinea pig hepatocytes by Ca-mobilizing agonists can be resolved into two phases. The initial transient phase is independent of extracellular Ca, and is probably a result of Ca released from an intracellular pool. The second phase occurs only in the presence of extracellular Ca, which suggests that Ca-influx is also involved in the mechanism of Ca-mobilization by these agents in the guinea pig hepatocyte. 相似文献
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To study whether absolute (m/s) or relative (body lengths/s) speed should be used to compare the vulnerability of differently sized animals, we developed a simple computer simulation. Human 'predators' were asked to 'catch' (mouse-click) prey of different sizes, moving at different speeds across a computer screen. Using the simulation, a prey's chances of escaping predation depended on its speed (faster prey were more difficult to catch than slower prey of the same body size), but also on its size (larger prey were easier to catch than smaller prey at the same speed). Catching time, the time needed to catch a prey, also depended on both prey speed and prey size. Relative prey speed (body lengths/s or body surface/s) was a better predictor of catching time than was absolute prey speed (m/s). Our experiment demonstrates that, in contrast to earlier assertions, per unit body length speed of prey may be more 'ecologically relevant' than absolute speed. Copyright 1998 The Association for the Study of Animal Behaviour. 相似文献
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Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. We employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF(L47M). The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. Our experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling. 相似文献
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We have used in vitro mutagenesis to introduce a six residue histidine sequence (His-tag) near the amino terminal end of the human PGHS-1 and -2 and have expressed these proteins using the baculovirus system. The His-tags are located one and two amino acids beyond the signal peptide cleavage sites of PGHS-1 and PGHS-2, respectively, positions that do not affect their activities or sensitivities to nonsteroidal anti-inflammatory drugs. When expressed in sf-21 cells, the His-tagged enzymes have K(m) values for arachidonate, and IC(50) values for inhibition by nonsteroidal anti-inflammatory drugs that are similar to values reported for the nontagged enzymes. The His-tags allowed for purification of the PGHSs by a simplified protocol involving nickel-affinity and anion exchange FPLC chromatography. The specific activities and recoveries for the purified enzymes were as good or better than those reported previously for purification of the non-tagged PGHS. These baculovirus constructs should provide a convenient source for pharmacologic and biophysical studies that require large scale preparation of human PGHSs. 相似文献
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DeWitt MA Kliegman JI Helmann JD Brennan RG Farrens DL Glasfeld A 《Journal of molecular biology》2007,365(5):1257-1265
The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 A. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR. 相似文献