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1.
The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.  相似文献   

2.
The polypeptides of mouse B16 melanoma lines of defined metastatic potential have been analyzed by two-dimensional electrophoresis. Parent B16 melanoma and two independently isolated B16-F1 lines, which are low metastatic, exhibited a new polypeptide, Ax (pI 5.2; Mr = 43,000), comprising approximately 30% of the total actin, in addition to normal beta- and gamma-actin. The Ax is present in the Triton-insoluble fraction (cytoskeleton and nuclear matrix) as well as in the Triton-soluble fraction at a constant ratio of about 0.5 to beta- plus gamma-actin. The Ax polypeptide has been identified as a variant form of actin by immunostaining with anti-actin antibody and by a comparison of its tryptic patterns with those produced by beta- and gamma-actin polypeptides; the Ax is also identified as a component of microfilaments. On the other hand, the Ax polypeptide disappears or its expression is very low in high metastatic lines, two independently isolated B16-F10s and B16-BL6. By in vitro translation, we have identified the mRNA species that code for Ax in B16-F1, but not in B16-F10.  相似文献   

3.
Posttranslational modifications and intracellular transport of the D2- cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A) and 131,000-158,000 (B) were synthesized using radiolabeled precursors in cultured neurons. A and B were found to contain only N-linked complex oligosaccharides, and both polypeptides appeared to be polysialated as determined by [14C]mannosamine incorporation and precipitation with anti-polysialic acid antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination by photoactivation employing a hydrophobic labeling reagent. In rat forebrain explant cultures, D2-CAM was synthesized as four polypeptides: A (195,000 Mr), B (137,000 Mr), C (115,000 Mr), and a group of polypeptides in the high molecular weight region (HMr) between 250,000 and 350,000. Peptide maps of the four polypeptides yielded similar patterns. Biosynthesis of C and HMr increased with age, relative to A and B. A and B were sulfated in embryonic brain, however, sulfation was not noticeable at postnatal ages. Phosphorylation, on the other hand, of A and B was observed at all ages examined. We suggest that D2-CAM function may be modified during development by changes in the relative synthesis of the different polypeptides, as well as by changes in their glycosylation and sulfation.  相似文献   

4.
The L1 cell adhesion molecule is involved in cell migration and cell-cell adhesion in the brain. In this report we describe a purification procedure which allowed simultaneous isolation of L1 and the neural cell adhesion molecule. Furthermore, we studied L1 biosynthesis, post-translational modifications and function. L1 was synthesized as a polypeptide with relative molecular weight 200,000 in monolayer cultures of fetal rat neurons and in explant cultures of rat forebrain. The L1 polypeptide was co-translationally inserted into the membranes of the endoplasmic reticulum as an integral membrane protein. Both sulfation and phosphorylation of L1 was observed. L1 polypeptides with lower relative molecular weight which were present on the plasma membrane were probably derived from the 200,000 mol. wt polypeptide. The function of L1 was investigated and an L1 involvement in neurite fasciculation was demonstrated.  相似文献   

5.
We have previously reported lack of expression of a polypeptide designated L3 in infant acute lymphoblastic leukemia (ALL). Expression of L3 occurred predominantly in older children with pre-B ALL. We have recently reported the expression during B cell ontogeny of two other polypeptides, designated L2 and L4 with a similar Mr as L3, which were identified as phosphorylated and non-phosphorylated forms respectively of the low Mr heat shock protein. hsp27. In this study we have characterized L3 and identified it as another phosphorylated form of hsp27. The two phosphorylated forms appear to be differentially expressed in acute leukemia. L3 levels in infants who expressed hsp27 isoforms L2 and L4 were significantly diminished compared to levels in older children with an equivalent amount of hsp27. We conclude that leukemic cells in infant ALL exhibit a unique pattern of phosphorylation of hsp27 expressed at a pre-B cell stage of differentiation.  相似文献   

6.
A monoclonal antibody obtained from mice immunized with a crude neurofilament preparation from newborn rat brain revealed the existence of heterogeneity of the 200,000- and 150,000-dalton neurofilament polypeptides. On immunoblot the monoclonal antibody iC8 reacted with both the 200,000- and 150,000-dalton components in the CNS, but only with the 150,000-dalton polypeptide in sciatic nerve preparations. In addition, the 150,000-dalton polypeptide appeared as a single band in the sciatic nerve, whereas in the CNS a doublet was labeled by iC8. In contrast a second monoclonal antibody (3H5) reacted with the 200,000-dalton peptide and a single 150,000-dalton component in both the central and peripheral nervous system preparations. The differences revealed by iC8 were probably not due to phosphorylation, as the pattern of antibody binding in immunoblots was not changed by pretreatment with alkaline phosphatase. The findings suggest that different isoforms of neurofilament polypeptides are present in the nervous system.  相似文献   

7.
Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.  相似文献   

8.
The polypeptides recognized by a monoclonal antibody against calf thymus DNA polymerase alpha (secreted from a hybridoma CL22 -2- 42B , Nucleic Acids Res. (1982) 10, 4703-4713) were identified by the immunoblot method as the large polypeptides of the partially-purified 10S DNA polymerase alpha fraction. Using an immunoprecipitation technique with the monoclonal antibody, a rapid immunological isolation of the polypeptides has been achieved. By this method, the large polypeptides with Mr = 140,000, 145,000, and 150,000 were isolated from a partially-purified preparation of 10S DNA polymerase alpha. On the other hand, the polypeptides with Mr = 150,000, 180,000, and 240,000 were obtained from a crude extract of calf thymus. Tryptic peptide maps showed that the large polypeptides with Mr = 150,000, and 180,000 were very similar in primary structure and that the structures of Mr = 180,000 and 240,000 polypeptides contained partially common sequences. Among these polypeptides, the Mr = 150,000 polypeptide was shown to correlate with the enzyme activity. These results suggest that the large polypeptide of 10S DNA polymerase alpha is initially synthesized as Mr = 180,000 or larger polypeptide, then converted to the form with Mr = 150,000. The Mr = 140,000 and 145,000 polypeptides in the purified preparation may be artificial products formed during purification.  相似文献   

9.
A high-Mr phosphoprotein (Mr 442,000) was purified from Nonidet-P-40-solubilized plasma membranes of cultured human skin fibroblasts. The protein comprised one 200,000-Mr subunit consisting of 116,000- and 84,000-Mr polypeptides and two identical 121,000-Mr subunits each consisting of 66,000- and 55,000-Mr polypeptides. The 200,000-Mr subunit and its polypeptides contained phosphotyrosine residues and were also [32P]phosphorylated at these residues from [gamma-32P]ATP in vitro by an intrinsic tyrosine kinase activity of the protein molecule in response to the presence of hyaluronate precursors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The 121,000-Mr subunits and their polypeptides contained phosphoserine residues that could not be [32P]phosphorylated during autophosphorylation of the protein in vitro. The protein molecules separated from exponential- and stationary-growth-phase cells were identical in their quaternary structure, but appeared to exist in different proportions with respect to the state of phosphorylation of their 121,000-Mr subunits during different growth phases of the cell. Phosphorylation of polypeptides appeared to predispose in favour of their UDP-glucuronic acid- and UDP-N-acetylglucosamine-binding activities. The phosphorylated 116,000- and 84,000-Mr polypeptides of 200,000-Mr subunits possessed a single binding site for UDP-glucuronic acid and UDP-N-acetylglucosamine respectively. The phosphorylated 200,000-Mr subunit could also cleave the UDP moiety from UDP-glucuronic acid and UDP-N-acetylglucosamine precursors. The phosphorylated 121,000-Mr subunit possessed two binding sites with equal affinity towards UDP-glucuronic acid and UDP-N-acetylglucosamine but did not possess UDP-moiety-cleavage activity. The phosphorylation of 200,000-Mr subunit by an intrinsic kinase activity of the protein molecule appeared to elicit its oligosaccharide-synthesizing activity, whereas phosphorylation of 121,000-Mr subunits, presumably carried out in vivo, abolished this activity of the protein molecule. The oligosaccharides synthesized by the protein were about Mr 5000 and about 12 disaccharide units in length. Neither nucleotide sugars nor glycosyl residues nor newly synthesized oligosaccharides were bound covalently to the protein molecule. The UDP moiety of nucleotide sugar precursors did not constitute a link between protein molecule and oligosaccharide during its synthesis. Although isolated 442,000-Mr protein did not synthesize high-Mr hyaluronate in vitro, this protein molecule can be considered as a constituent of membrane-bound hyaluronate synthase complex because of its observed properties.  相似文献   

10.
Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.  相似文献   

11.
Stimulation of platelets by thrombin causes an increase in the amount of cytoskeleton proteins insoluble in 1% Triton X-100, i.e. myosin, actin, actin-binding protein, an alpha-actinin-like protein of Mr = 105,000, unidentified polypeptides of Mr = 150,000, 31,00, and under some conditions, 56,000. Concurrently the Mr = 20,000 light chains of myosin and a cytoplasmic Mr = 42,000 polypeptide are phosphorylated, presumably by calmodulin-Ca2+-dependent myosin light chain kinase and a phospholipid-Ca2+-dependent kinase, respectively. The adenylate cyclase stimulators prostaglandin D2 (PGD2) and forskolin increased platelet cyclic AMP and prevented the phosphorylation of these polypeptides and the increase in Triton-insoluble cytoskeleton proteins. When added to platelets after stimulation by thrombin they caused rapid complete reversal of myosin light chain and Mr = 42,000 polypeptide phosphorylation; simultaneously the association of myosin with the cytoskeleton proteins and the increase in the content of each of the Triton-insoluble cytoskeleton proteins (except the Mr = 56,000 polypeptide) was reversed. The amount of Triton-insoluble myosin was affected more readily by PGD2 or forskolin than were the other proteins. Increasing thrombin from 0.1 to 1.0 unit/ml inhibited all the responses to PGD2 and forskolin possibly due to concentration-dependent effects of thrombin that inhibit adenylate cyclase. These results suggest that cytoskeleton assembly and activation of the contractile apparatus in intact platelets are readily reversible by cyclic AMP-dependent reactions.  相似文献   

12.
The mechanism of tumor promotion is not well understood. We have used the transformable, tumor promotable, mouse embryo fibroblast C3H/10T1/2 Cl 8 cells to study tumor promoter specific changes in protein synthesis and protein glycosylation. Two-dimensional gel electrophoresis showed that 12-O-tetradecanoylphorbol 13-acetate caused a significant increase in the synthesis of five cellular and 34 extracellular polypeptides. One of these polypeptides has tentatively been identified as ornithine decarboxylase. One new polypeptide (p 62, Mr 58,000) was found in the medium of 12-O-tetradecanoylphorbol 13-acetate-treated cells. The amounts of several excreted proteins were enhanced 5-10 fold by 12-O-tetradecanoylphorbol 13-acetate. 12-O-tetradecanoylphorbol 13-acetate interfered with glycosylation both by affecting protein synthesis and also directly with glycosylation. At least 15 polypeptides in the medium and two cellular polypeptides decreased after 12-O-tetradecanoylphorbol 13-acetate treatment. Two of the major polypeptides found in the medium (p 8 and 10, Mr approx. 200,000-220,000) have properties similar to fibronectin, while p 9 and 11 both found in the cellular preparations and in the medium (Mr 180,000 and 150,000) were collagenase sensitive and their synthesis was inhibited by 12-O-tetradecanoylphorbol 13-acetate.  相似文献   

13.
N-CAM from rat brain consists of three polypeptides: 190,000 Mr (A), 140,000 Mr (B) and 120,000 Mr (C). It has been reported that cultured neurons express only A and B, whereas glial cultures synthesize mainly B and C. During postnatal development the relative biosynthesis of C increases. This could possibly reflect differentiation of neurons or an increased biosynthetic contribution of glial cells. We have investigated neuronal expression of N-CAM with the aim of determining whether neurons were able to synthesize the C-polypeptide. Biosynthetic labelling of explant cultures of peripheral ganglia and of chromaffin cells from adrenal medulla showed that cultured neurons synthesized not only A and B, but also C. However, the biosynthetic capacity for C production was low. Cell-free translation of microsomes from neuronal cell cultures showed that they contained a messenger RNA coding for C. Finally, retinal ganglion neurons expressed C when located in their natural environment as determined by biosynthetic labelling performed in living rats. Thus, both neurons and glial cells may be involved in the developmentally regulated change in C expression that occurs during postnatal life.  相似文献   

14.
The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) consist of two polypeptides each belonging to a new class of molecules referred to as the hemopoietin receptor family. When expressed alone, receptor polypeptides of this family often bind their respective factors with lower affinity than the receptors identified in whole cells. Despite the lack of structural evidence for any enzymatic activity of the receptor polypeptides, both IL-3 and GM-CSF stimulate tyrosine phosphorylation of multiple intracellular substrates. We investigated IL-3 and GM-CSF receptor structure and signaling in a myeloid cell line, FDC-P1, which is dependent on either IL-3 or GM-CSF for growth. Antiphosphotyrosine antibodies were used to immunoprecipitate tyrosine-phosphorylated proteins from 32P-labeled cells or to probe immunoblots. Both IL-3 and GM-CSF stimulated the phosphorylation of a similar pattern of polypeptides on tyrosine. One tyrosine phosphorylated polypeptide migrated with M(r) = 135,000 and increased to 150,000 over a period of 10 min following stimulation of cells with IL-3 or GM-CSF. The M(r) = 135,000-150,000 polypeptide phosphorylated in response to IL-3 was shown to be primarily the Aic-2A polypeptide, the low affinity IL-3 receptor. Phosphatase treatment showed that the dramatic IL-3-induced shift in apparent molecular weight from M(r) = 125,000 in unstimulated cells was entirely due to phosphorylation. The closely related receptor, Aic-2B, was also tyrosine phosphorylated in response to IL-3, although to a lesser extent than Aic-2A. Treatment with GM-CSF resulted in tyrosine phosphorylation of the Aic-2B polypeptide exclusively. It was intriguing that GM-CSF treatment did affect the mobility of the Aic-2A polypeptide on polyacrylamide gels. Together, these results suggest that the Aic-2A polypeptide is part of the IL-3 receptor complex, but not the GM-CSF receptor. In contrast, the Aic-2B polypeptide is a component of the GM-CSF receptor, but it can also be utilized in an IL-3 receptor.  相似文献   

15.
Two cell surface molecules found in mouse brain, N-CAM and the L1 antigen, were compared in terms of their cell adhesion function, polypeptide structures, antigenic determinants and distribution in cerebellar tissue. Fab fragments of polyclonal antibodies to either N-CAM or L1 antigen only partially inhibited the rate of calcium-independent aggregation of neuroblastoma N2A cells, whereas complete and more efficient inhibition was obtained when they were used in combination. Despite the functional similarity, comparison of the electrophoretic behaviour of the purified molecules and of their proteolytic fragments shows that the L1 antigen polypeptide is distinct from that of N-CAM. In addition, no antigenic cross-reactivity was detected between the two molecules. In cryostat sections of cerebellum from young post-natal mice, N-CAM was found to be present in all cell and neurite layers, whereas L1 antigen was expressed only in regions containing post-mitotic cells. These results indicate that two chemically and histochemically distinct cell surface polypeptides can contribute to the calcium-independent adhesiveness of neural cells, and suggest that their differential expression might cause adhesive specificity among cells of developing neural tissues.  相似文献   

16.
17.
Urokinase (u-PA) proteolytically cleaves both human plasma (pFn) and cellular (cFn) dimeric fibronectin (M(r) 440,000) into four major polypeptides of approximately M(r) 210,000, 200,000, 25,000, and 6,000. Amino acid sequence analysis of the polypeptide fragments indicated that the enzymatic cleavage of Fn occurs at two sites: 1) between an arginine/alanine peptide bond located C-terminal to residue 259; this cleavage liberates the N-terminal M(r) 25,000 fragment and the M(r) 210,000 and M(r) 200,000 polypeptides derived from the A and B chains of Fn, respectively; and 2) between an arginine/threonine peptide bond located C-terminal to residue 2,299, thereby yielding an M(r) 6,000 dimeric fragment containing the C-terminal interchain disulfide bonds. Predigestion of Fn with u-PA increased the molecule's vulnerability to further attack by the enzymes plasmin and cathepsin D. These data provide further biochemical evidence for the proteolytic cleavage of fibronectin by plasminogen activators and substantiate that u-PA digestion of Fn may be an initial event in the local degradation of the extracellular matrix by malignant cells, possessing elevated levels of these enzymes.  相似文献   

18.
19.
A monospecific rabbit antiserum to pepsin-extracted chick gizzard type VI collagen was used to characterize the intact forms of type VI collagen in tissues and cultured cells. Immunoblotting of gizzard extracts revealed polypeptides of Mr ranging from 260,000 to 140,000. Components of about Mr = 260,000, 150,000, and 140,000, each with a different peptide profile, were immunoprecipitated from labeled matrix-free chick embryo cells. Cleavage of the immunoprecipitated polypeptides with pepsin generated pepsin-resistant fragments of about Mr = 70,000, 55,000, and 45,000 that represent the alpha 1(VI), alpha 2(VI), and alpha 3 (VI) fragments. Immunoblotting with affinity-purified antibodies indicated that the Mr = 150,000 is the intact parent polypeptide of the alpha 1(VI) pepsin; the Mr = 140,000 of the alpha 2(VI) pepsin, and the Mr = 260,000 of the alpha 3(VI) pepsin. Association of the three parent chains was studied by pulse-chase experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under nonreduced conditions. A complex of Mr = 500,000 is already present intracellularly at the end of a 7-min pulse and increases considerably with time while the three unassembled chains show a comparable decrease. After 5-15 min of chase larger forms appeared along with small amounts of aggregated material that did not enter the gel. Analysis of the immunoprecipitate by diagonal electrophoresis indicated that the component of Mr = 500,000 and the larger forms dissociated into the Mr = 260,000, 150,000, and 140,000 polypeptides. Sedimentation profile of a labeled cell extract on a 5-20% sucrose gradient under nondenaturing conditions confirmed the presence of three different peptides in the complex.  相似文献   

20.
A monoclonal antibody, mAb 1A, that immunoprecipitates the [3H]PN200-110-binding complex from rabbit skeletal muscle has been used to study the subunit structure of the dihydropyridine-sensitive, voltage-activated calcium channel. Digitonin-solubilized [3H]PN200-110-binding component, purified by wheat germ agglutinin chromatography, sediments as a 21 S complex. The sedimentation coefficient of the complex is increased to about 24 S after incubation with mAb 1A IgG. Four polypeptides with apparent molecular weights under nonreducing conditions of 220,000, 200,000, 61,000, and 33,000 co-sediment with the 21 S complex. mAb 1A recognizes the Mr 200,000 polypeptide, as shown by Western blotting analysis. [3H] PN200-110 complex purified by wheat germ agglutinin chromatography followed by immunoaffinity chromatography on an mAb 1A column is comprised primarily of the same four polypeptides. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the Mr 220,000 protein migrates as a polypeptide of Mr 143,000; the mobility of the Mr 200,000 protein recognized by mAb 1A is unaffected by reduction. Thus, the Mr 200,000 polypeptide appears to be a previously undescribed component of the dihydropyridine-binding complex and, in association with the other polypeptides, may comprise the voltage-sensitive calcium channel.  相似文献   

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