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1.
Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.  相似文献   
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Flower buds of `Valencia' orange (Citrus sinensis [L.] Osbeck) were able to fix 14CO2 into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total 14C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the 14C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO2 per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO2 into different compounds suggests that this CO2 assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting.  相似文献   
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The phylogenetic distributions ofσ- and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [3H]haloperidol and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine, ligands that bind with high affinity to σ- and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [3H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [3H]N-[1-(2-thienyl)cyclohexyl]-3,4–piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that σ– and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.  相似文献   
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Human melanoma cells express a novel integrin receptor for laminin   总被引:11,自引:0,他引:11  
This study sought to determine whether human melanoma cells express integrin-related receptors that mediate their adhesion to laminin. We found that antibodies against the integrin beta 1 chain blocked cell attachment to laminin-coated surfaces. Furthermore, immunofluorescence staining demonstrated beta 1 complexes in vinculin-positive focal adhesion plaques on the basal surface of cells attached to laminin substrates. Chromatography of detergent extracts of 125I-surface-labeled cells on laminin-Sepharose columns recovered two major laminin-binding proteins (100 and 130 kDa, reduced) that bound with high affinity to the columns and were eluted with EDTA. Both proteins were specifically immunoprecipitated from column fractions with monoclonal and polyclonal antibodies to the integrin beta 1 subunit, indicating that they form a noncovalent heterodimer complex. The alpha-like subunit is composed of a 30-kDa light chain that is joined by a disulfide bond to the 100-kDa heavy chain. This complex was not recovered from columns of fibronectin- or collagen type I- or IV-Sepharose. Laminin-binding by the alpha beta 1 complex was independent of Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg-like sequences, but required the presence of divalent cations. The 100-kDa alpha-like subunit was electrophoretically and immunochemically distinct from the other known alpha subunits, alpha 1-alpha 6. The results indicate that human melanoma cells express a novel laminin-specific integrin beta 1 complex which may mediate the cells' interactions with this ligand.  相似文献   
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Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.  相似文献   
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We studied the effects of H2O/D2O substitution on the permeation and gating of the large conductance Ca2+-activated K+ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K+>Rb+≫Li+, Na+, Cs+ and Cl. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D2O as compared to H2O. The blockade of channel conductance by cytosolic Ca2+ weakened in D2O as a result of a decrease in zero voltage Ca2+ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D2O primarily due to the change in Ca2+ binding to the channel during the activation step. The Hill coefficient for Ca2+ binding was 3 in D2O and around 1 in H2O. The values of the Ca2+ binding constant in the open channel conformation were 0.6 and 6 μm in H2O and D2O, respectively, while the binding in the closed conformation was much less affected by D2O. The H2O/D2O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca2+.  相似文献   
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Accurate retention time (RT) prediction is important for spectral library-based analysis in data-independent acquisition mass spectrometry-based proteomics. The deep learning approach has demonstrated superior performance over traditional machine learning methods for this purpose. The transformer architecture is a recent development in deep learning that delivers state-of-the-art performance in many fields such as natural language processing, computer vision, and biology. We assess the performance of the transformer architecture for RT prediction using datasets from five deep learning models Prosit, DeepDIA, AutoRT, DeepPhospho, and AlphaPeptDeep. The experimental results on holdout datasets and independent datasets exhibit state-of-the-art performance of the transformer architecture. The software and evaluation datasets are publicly available for future development in the field.  相似文献   
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