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1.
A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.  相似文献
2.
Signal transduction by receptors with tyrosine kinase activity   总被引:501,自引:0,他引:501  
A Ullrich  J Schlessinger 《Cell》1990,61(2):203-212
3.
Gene regulation by steroid hormones   总被引:329,自引:0,他引:329  
M Beato 《Cell》1989,56(3):335-344
4.
Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior.  相似文献
5.
P Chavrier  R G Parton  H P Hauri  K Simons  M Zerial 《Cell》1990,62(2):317-329
A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library. We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies. One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus. The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes. These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.  相似文献
6.
7.
The mannose 6-phosphate receptor and the biogenesis of lysosomes   总被引:122,自引:0,他引:122  
Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.  相似文献
8.
9.
rab5 controls early endosome fusion in vitro   总被引:110,自引:0,他引:110  
J P Gorvel  P Chavrier  M Zerial  J Gruenberg 《Cell》1991,64(5):915-925
The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab5lle133, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion.  相似文献
10.
We have examined the interaction between the platelet-derived growth factor (PDGF) receptor and three src family tyrosine kinases, pp60c-src, p59fyn, and pp62c-yes. The kinase activities of all three enzymes were elevated after PDGF stimulation of quiescent fibroblasts, coincident with association of the src family kinases with the PDGF receptor and other proteins. The presence of a protein of 81-85 kd in these complexes correlated with the detection of phosphatidylinositol (PI) kinase activity (previously described to associate with both the PDGF receptor and pp60c-src-middle T antigen). These results suggest that the physiological response to PDGF involves interaction of the receptor not only with serine/threonine and lipid kinases and a phospholipase, but also with other tyrosine kinases.  相似文献
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