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31.
Differential expression of two related amino acid transporters with differing substrate specificity in Arabidopsis thaliana 总被引:8,自引:3,他引:5
Marion Kwart Brigitte Hirner Sabine Hummel Wolf B. Frommer 《The Plant journal : for cell and molecular biology》1993,4(6):993-1002
A general amino acid permease cDNA ( AAP2 ) was isolated from Arabidopsis by complementation of a yeast mutant defective in citrulline uptake. Direct transport measurements in yeast show that the protein mediates uptake of l -[14 C]-citrulline and l -[14 C]-proline. Detailed analyses of the substrate specificity by competition studies demonstrate that all proteogenic amino acids are recognized by the carrier, including those that represent the major transport forms of reduced nitrogen in many species, i.e. glutamine, glutamate and asparagine. Thus, AAP2 is less selective as compared with AAP1 and transports basic amino acids such as histidine as shown by expression in a histidine transport-deficient yeast strain. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows significant homologies to the Arabidopsis broad specificity permease AAP1, and a limited homology to bacterial branched chain amino acid transporters, but not to any other known proteins. Alterations in the charged residues as compared with AAP1 in four regions might be involved in the difference in selectivity towards basic amino acids. Both genes are highly expressed in developing pods indicating a role in supplying the developing seeds with reduced nitrogen. AAP2 is selectively expressed in the stem and might therefore play a role in xylem-to-phloem transfer of amino acids during seed filling. Furthermore in situ hybridization shows that both genes are expressed in the vascular system of cotyledons in developing seedlings. 相似文献
32.
Kinematic characteristics of the fore- and hindlimb displacements during terrestrial and aquatic locomotions in juvenile marine turtles Dermochelys coriacea are compared. Modulations of the spatial displacements of the limbs and durations of the stance and swing phases are analysed in relationship with the constraints of the aquatic and terrestrial environments. The stance and swing phases used for describing the aquatic locomotion are re-evaluated in the light of the spatial displacements of the forelimbs during complete beating cycles. 相似文献
33.
Klaus Werner Wolf Sabine Glatzel Frauke Niedereichholz Bryan M. Turner 《Biotechnic & histochemistry》1996,71(3):137-144
We describe a method for isolating chromosomes from testes of the desert locust, Schistocerca gregaria, and their subsequent incubation with antibodies directed against chromosomal proteins. The procedure involves hypotonic pretreatment of the germ cells, centrifugation onto coverslips in a cytocentrifuge and immunolabeling, while still unfixed, using a chromatin-stabilizing buffer. In the present case, an antibody specific for the acetylated isoforms of his tone H4 was tested. After the antibody treatment, the preparations are fixed using formaldehyde, stained with a DNA-specific fluorescent dye and mounted. Analysis of the preparations revealed good preservation of chromosome structure in prophase spermatogonia and late prophase I spermatocytes. Fully condensed chromosomes were not observed and are probably lost during preparation. The bright fluorescence of the autosomes indicates that the reaction between the antibody against acetylated histone H4 and its chromosomal antigen is not impeded. In contrast, the X univalent remained unstained with the exception of a small terminal band. Thus, cytospin preparations of locust germ cells allow high resolution immunolabeling with antibodies against chromosome-associated proteins. 相似文献
34.
A new method was developed to purify the streptococcal hyaluronate synthase in active form to electrophoretic homogeneity. The method is based on the extraction of protoplast membranes with digitonin and a phase separation into an aqueous and a detergent phase induced by addition of polyethylene glycol 6000 at 0°C. Proteins bound to hyaluronate were enriched in the aqueous phase, whereas other membrane proteins resided in the detergent phase. Final purification of the hyaluronate synthase was achieved by ion exchange chromatography. 相似文献
35.
Sabine Lhernould Yannis Karamanos Patrice Lerouge Henri Morvan 《Glycoconjugate journal》1995,12(1):94-98
The peptide-N
4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992)Glycoconjugate J
9:191–97] was partially purified from culturedSilene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of unconjugated N-glycans in a suspension medium of culturedSilene alba cells.Abbreviations GlcNAc
N-acetylglucosamine
- PNGase
peptide-N
4-(N-acetylglucosaminyl) asparagine amidase
- BSA
bovine serum albumin
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TLC
thin layer chromatography
- HPAEC-PAD
High pH anion exchange chromatography-pulsed amperometric detection 相似文献
36.
Sabine Castano Bernard Desbat Isabelle Cornut Philippe Méléard Jean Dufourcq 《Letters in Peptide Science》1997,4(4-6):195-200
De novo designed extremely simplified amphipathic basicLeuiLysj (i = 2j) peptides of 8, 9 and 15residues were synthesized to clarify the mechanism of action of naturalcytotoxic and hemolytic small proteins or peptides. They proved to havestrong hemolytic activity towards human erythrocytes which increases withpeptide length. These peptides are highly surface active and form stablepeptidic films at the air/water interface. The sensitive and efficient FTIRmodulated polarization technique (PMIRRAS) allows one to obtain in situstructural and orientational information about the peptides at theinterface. A transition of secondary structure is observed: the shorterpeptides (8 and 9 residues) adopt -sheet structures while the longerone (15 residues) is folded into an -helix. In both cases, the peptideslie with the axis parallel to the interface. Their insertion into adimyristoylphosphatidylcholine monolayer can be followed from the increasein the surface and/or pressure of the films. In the mixed films, thepeptides adopt the same structure and orientation as observed at theair/water interface. Therefore, among the same series of peptides, atransition from -sheet to -helix occurs when the length increases(roughly >10 aa), but despite this drastic change both types ofstructures result in strongly hemolytic peptides. 相似文献
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Klaus Kayser Sabine André Gerhard Böhm Sonia Donaldo-Jacinto Peter Fritz Herbert Kaltner Gian Kayser Wolf-Peter Kunze Andreas Nehrlich Fu-Yue Zeng Hans-Joachim Gabius 《Development genes and evolution》1995,204(5):344-349
Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs. 相似文献