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排序方式: 共有234条查询结果,搜索用时 15 毫秒
41.
42.
G Semenza 《FEBS letters》2001,509(3):343-344
43.
J Ruf H Wacker P James M Maffia P Seiler G Galand A von Kieckebusch G Semenza N Matei 《The Journal of biological chemistry》1990,265(25):15034-15039
alpha,alpha-Trehalase (EC 3.2.1.28), an intrinsic protein of intestinal brush-border membranes, was purified to homogeneity from rabbits. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequence of a CNBr peptide were employed in a polymerase chain reaction to amplify a 71-base pair fragment of trehalase DNA with rabbit intestine cDNA as a starting template. This fragment was used as a hybridization probe to isolate full length trehalase clones from a rabbit intestine cDNA bank. Sequence analysis revealed that trehalase comprises 578 amino acids, contains at the amino terminus a typical cleavable signal sequence, at the carboxyl terminus a rather hydrophobic region typical of proteins anchored via glycosylphosphatidylinositol, and four potential N-glycosylation sites. Trehalase has no sequence homologies with other sequenced brush-border glycosidases. Northern blot analysis revealed a 1.9-kilobase trehalase mRNA in small intestine and kidney, smaller amounts in liver, and none in lung. Southern blot analysis indicated the gene has a length of 20 kilobase pairs or less. Injection into Xenopus laevis oocytes of mRNA synthesized in vitro from a trehalase template resulted in the expression of trehalase activity several hundredfold above background. The trehalase activity was membrane-bound and could be solubilized upon digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. This strongly suggests that rabbit small intestinal trehalase is anchored via glycosylphosphatidylinositol also when expressed in X. laevis oocytes. 相似文献
44.
ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway 总被引:71,自引:0,他引:71
Resident proteins of the ER lumen carry a specific tetrapeptide signal (KDEL or HDEL) that prevents their secretion. We have previously described the isolation of yeast mutants that fail to retain such resident proteins within the cell. Here we describe ERD2, a gene required for retention. It encodes a 26 kd integral membrane protein whose abundance determines the efficiency and capacity of the retention system. Reduced expression of ERD2 leads to secretion of proteins bearing the HDEL signal, whereas overexpression of ERD2 improves retention both in wild-type cells and in other mutants. These results are consistent with other evidence that ERD2 encodes the HDEL receptor (see accompanying paper). The gene is also required, perhaps indirectly, for normal protein transport through the Golgi, and hence for growth. We discuss possible roles for ERD2 in the secretory pathway. 相似文献
45.
Konrad Honold Barbara Ludeke Hans Hengartner Giorgio Semenza 《The Journal of membrane biology》1988,105(2):165-175
Summary The small intestinal brush border membrane is endowed with a number of transport systems. Monoclonal antibodies were produced against integral membrane proteins and tested for their ability to bind to such membranes. For this purpose papain-digested, deoxycholate-extracted BBMVs from rabbit small intestine were used to immunize mice. Of the 765 hybridoma supernatants tested, 119 gave a significantly higher extent of binding to the crude antigen preparation as compared with the background. The monoclonal antibodies were also tested for their ability to influence the sodium-dependent uptake of solutes into intact BBMVs. Two monoclonal antibodies clearly showed stimulation of secondary actived-glucose transport, whereas sodium-dependent uptake ofl-alanine andl-proline was not affected. Hydrophobically labeled, i.e. intrinsic, membrane proteins of 175, 78 and 65 kilodaltons could be immunoprecipitated by both monoclonal antibodies, the 78 kDa band corresponding in all likelihood to the Na+/glucose cotransporter. 相似文献
46.
Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane 总被引:1,自引:0,他引:1
Reconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein-depleted ghosts, or detergent-treated ghosts. D-glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein-depleted ghosts, the selective solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC-720 extracts could be prepared in which the glucose transporter retained activity for days at 4 degrees C. These extracts were applied to affinity chromatography matrices of phloretin-Agarose, prepared by coupling phloretinyl-3'-benzylamine (PBA) to CH-Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4.5, from the Affigel affinity medium. 相似文献
47.
cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme. 总被引:1,自引:0,他引:1 下载免费PDF全文
E Rached N M Hooper P James G Semenza A J Turner N Mantei 《The Biochemical journal》1990,271(3):755-760
Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form. 相似文献
48.
Cell-free synthesis, membrane integration, and glycosylation of pro-sucrase-isomaltase 总被引:4,自引:0,他引:4
Cell-free translation of total RNA from rabbit intestinal mucosa in a rabbit reticulocyte lysate, after immunoprecipitation with antibodies directed against sucrase-isomaltase, yielded a polypeptide of 200 kDa, which was identified as pro-sucrase-isomaltase. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of an additional 220-kDa polypeptide. The 220-kDa polypeptide was associated with the membranes in a way that made it inaccessible to proteolysis; this protection was abolished by lytic detergent concentrations, indicating that the polypeptide was segregated into the microsomal vesicle. The 220-kDa polypeptide was glycosylated as evidenced by it being bound to concanavalin A-Sepharose and eluted with alpha-methyl-D-mannopyranoside. The increase in apparent molecular mass (approximately 20 kDa) of the primary translation product upon translocation was due to the addition of carbohydrate; treatment of the 220-kDa polypeptide with endo-beta-N-acetylglucosaminidase H increased its electrophoretic mobility to that of the 200-kDa polypeptide which was obtained in the absence of membranes. Partial N-terminal amino acid sequence of a translation product labeled with [3H]Leu in the absence of membranes revealed that Leu was incorporated into identical positions as in the final (pro)-sucrase-isomaltase, thus indicating the lack of a transient signal peptide. 相似文献
49.
50.
We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na+ and d-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27–56). We present here a study on the inactivation of the Na+-dependent transport systems, but not of the Na+-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na+ was observed, the parallelism between Na+-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner ((1986) J. Biol. Chem. 261, 1041–1047) in the sugar binding site of the renal Na+/d-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule. 相似文献