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41.
Steroid binding activity is retained in a 16-kDa fragment of the steroid binding domain of rat glucocorticoid receptors 总被引:4,自引:0,他引:4
S S Simons F D Sistare P K Chakraborti 《The Journal of biological chemistry》1989,264(24):14493-14497
The steroid binding domain of the rat glucocorticoid receptor is considered as extending from amino acids 550 to 795. However, such a synthetic protein (i.e. amino acids 547-795; Mr approximately 31,000) has been reported to show very little affinity for the potent synthetic glucocorticoid dexamethasone. We now disclose that digestion of steroid-free rat glucocorticoid receptors with low concentrations of trypsin yields a single species, of Mr = 16,000, that is specifically labeled by dexamethasone 21-mesylate. This 16-kDa fragment retains high affinity binding for [3H]dexamethasone that is only approximately 23-fold lower than that seen with the intact 98-kDa receptor. Analysis of the protease digestion patterns obtained both with trypsin and with lysylendopeptidase C allowed us to deduce the proteolytic cleavage maps of the receptor with these enzymes. From these protease maps, the sequence of the 16-kDa fragment was identified as being threonine 537 to arginine 673. These results show that glucocorticoid receptor fragments smaller than 34 kDa do bind steroids and that the amino acids Thr537-Arg673 constitute a core sequence for ligand binding within the larger steroid binding domain. The much slower kinetics in generating the 16-kDa fragment from affinity-labeled receptors suggests that steroid binding causes a conformation change in the receptor near the cleavage sites. 相似文献
42.
The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and -amino-N. These N-reserves enable an autonomous development of the seedling for 8–10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake system (vmax = 0.8 mol NO
3
-
·(g root FW)–1·h–1; Ks = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (vmax = 3.4 mol NO
3
-
·(g root FW)–1·h–1; Ks = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates of the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (t1/2 of vmax = 5 d) probably caused by the decay of carrier molecules. Small differences in Ks but significant differences in vmax indicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules.Abbreviations NR
nitrate reductase
-
pFPA
para-fluorophenylalanine
This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck. 相似文献
43.
44.
45.
Felix Gisela; Altmann Thomas; Uwer Ursula; Jessop Alison; Willmitzer Lothar; Morris Peter-Christian 《Journal of experimental botany》1996,47(8):1007-1017
A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant 相似文献
46.
Across the world there is a prevailing view that freshwater algae are cosmopolitan. The notion has seldom been tested and
is unlikely to be true in genetic terms. Nonetheless, some morphospecies of several groups of algae do have a worldwide distribution.
Others have restricted distributions and may be regarded as endemic to a region. However there is always the possibility that
they will be discovered in far away places. Australia has a rather large element of endemicity in its algal flora. From the
early days of Australian phycology many new genera and species of freshwater algae have been described. Some are of such distinctive
appearance or novelty as to be regarded as ‘flagship’ taxa. There is little doubt about their endemicity and their existence
increases the probability of less-distinguished species also being endemic. The degree of endemicity is probably masked by
the ‘force-fitting’ of European names to Australian species.
Some Australian endemics are robust and are widely distributed in a variety of types of water body. Others, the frail endemics,
the ones of greatest novelty and phylogenetic significance, have a very restricted range with their strongholds in dystrophic
coastal lagoons where tracts or remnant patches of native vegetation survive. Their survival and the conservation of their
biodiversity depends on recognition of the significance of coastal lagoons and swamps. 相似文献
47.
N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin. 总被引:7,自引:0,他引:7
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Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes. 相似文献
48.
The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein. 总被引:7,自引:0,他引:7
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P J Tienari B De Strooper E Ikonen M Simons A Weidemann C Czech T Hartmann N Ida G Multhaup C L Masters F Van Leuven K Beyreuther C G Dotti 《The EMBO journal》1996,15(19):5218-5229
We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP. 相似文献
49.
Socorro Durán Luis Sánchez-Linares Alejandro Huerta-Saquero Gisela Du Pont Alejandra Huerta-Zepeda Jorge Calderón 《Biochemical genetics》1996,34(11-12):453-465
We present evidence thatRhizobium etli has two glutaminases differentiated by their thermostability and electrophoretic mobility. The thermostable glutaminase (B) is constitutive, in contrast with the thermolabile glutaminase (A), which is positively regulated by glutamine and negatively regulated by ammonium and by the carbon source. In distinction to glutaminase A, glutaminase B plays a minor role in the utilization of glutamine as a carbon source, but it may play a role in maintaining the balance of glutamine and glutamate. By complementation of theRhizobium etli LM16 mutant that lacks glutaminase A, we have cloned the gene that codes for this enzyme. 相似文献
50.
Thomas Altmann Gisela Felix Alison Jessop Annette Kauschmann Ursula Uwer Hugo Peña-Cortés Lothar Willmitzer 《Molecular genetics and genomics : MGG》1995,247(5):646-652
Using a two-component Ac/Ds system consisting of a stabilized Ac element (Acc1) and a non-autonomous element (DsA), 650 families of plants carrying independent germinal DsA excisions/transpositions were isolated. Progenies of 559 of these Acc1/DsA families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel DsA fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for DsA insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for DsA insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the DsA insertion could be separated from the mutant locus by genetic recombination. 相似文献