首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
We examined the role of N-linked glycosylation of the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (Man-6-P) receptor in binding of [125I]IGF-II to the receptor. First we studied the synthesis and posttranslational processing of this receptor in rat C6 glial cells, which have abundant IGF-II/Man-6-P receptors. Cells were pulse labeled with [35S]methionine and lysed, and the IGF-II/Man-6-P receptor was immunoprecipitated using a specific IGF-II/Man-6-P receptor antibody (no. 3637). Analysis of the immunoprecipitate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reduction of disulfide bonds showed a 235-kDa receptor precursor that was processed into the mature 245-kDa IGF-II/Man-6-P receptor within 2 h of chase. Digestion of the 235-kDa precursor with endoglycosidase-H (Endo H) produced a 220-kDa form, whereas the mature 245-kDa receptor was relatively resistant to cleavage by Endo H. When cells were cultured in the presence of 2 microM monensin, the 235-kDa receptor was not further processed into the mature Endo H-resistant receptor form. In addition, the presence of swainsonine in C6 glial cell cultures led to the formation of a 240-kDa receptor hybrid molecule, which was cleaved by Endo H into a 225-kDa species. When tunicamycin was present during the pulse-chase labeling experiment, a 220-kDa receptor species accumulated. This species was 205 kDa by immunoblotting when SDS-PAGE was performed under nonreducing conditions. Pure IGF-II/Man-6-P receptor was digested with N-glycosidase-F, and the digest was immunoblotted with antiserum 3637 after SDS-PAGE under nonreducing conditions. Whereas undigested receptor was a single band of 215 kDa under nonreducing conditions, digested receptor was 205 kDa. The binding affinity of IGF-II for the digested receptor was the same as the binding affinity of IGF-II for the undigested receptor. In addition, affinity cross-linking experiments showed that [125I]IGF-II also bound to the unglycosylated receptor precursor that accumulated in the tunicamycin-treated cells, and the binding affinity of IGF-II for this species was indistinguishable from the binding affinity of IGF-II for the mature receptor. We conclude that IGF-II can bind to an IGF-II/Man-6-P receptor that lacks N-linked oligosaccharides.  相似文献   

2.
Studies of platelet-derived growth factor (PDGF) receptor biosynthesis and degradation have been limited by the lack of anti-receptor antibodies. In this study, peptides based on the cDNA-predicted amino acid sequence of the PDGF receptor were used to produce antisera that specifically immunoprecipitated the receptor. PDGF receptor biosynthesis was examined by pulse-chase labeling of cultured fibroblasts with [35S]methionine followed by immunoprecipitation. In BALB/c 3T3 fibroblasts the receptor was synthesized as a 160-kDa precursor that was converted to a mature 180-kDa form within 30-45 min. Removal of high mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent molecular weight of the 160-kDa precursor but did not affect the migration of the 180-kDa mature receptor. When mannosidase II was inhibited by swainsonine, the 160-kDa precursor failed to mature; instead a 168-kDa form of the receptor was observed. Nevertheless, swainsonine-treated cells responded mitogenically to PDGF. The mature 180-kDa form of the receptor had a half-life of approximately 3 h in the absence of ligand. Addition of PDGF reduced the receptor half-life to 45 min. These studies define and characterize a PDGF receptor precursor, show that receptor degradation is enhanced by PDGF, and demonstrate the functional integrity of incompletely processed PDGF receptors.  相似文献   

3.
The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].  相似文献   

4.
We have studied the biosynthesis of the cation-dependent mannose 6-phosphate receptor in murine BW5147 lymphoma cells and MOPC 315 plasmacytoma cells. The cells were labeled with [35S]methionine or [2-3H]mannose and the receptor immunoprecipitated with an anti-receptor antiserum. The receptor was first detected as a glycoprotein with an apparent molecular mass of 40 kDa. This intermediate was rapidly processed to a mature form which was stable during 22 h of chase. In these cells, the mature receptor has an apparent molecular mass of 43 kDa. The 3-kDa increase occurs as a result of processing of Asn-linked high-mannose oligosaccharides to complex-type units.  相似文献   

5.
A procedure was developed for the immunoprecipitation of glycosylated and nonglycosylated forms of the insulin receptor and its precursors without prior purification using lectins. 3T3-L1 adipocytes were labeled with [35S]methionine after which 35S-labeled receptor polypeptides were specifically immunoprecipitated and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The first 35S-polypeptide detected was a 190-kDa glycosylated proreceptor which was rapidly (t1/2 approximately equal to 15 min) processed to a 210-kDa intermediate. The latter precursor was more slowly (t1/2 approximately equal to 2 h) proteolytically processed to 125-kDa (alpha') and 83-kDa (beta') precursors of the mature alpha- and beta-receptor subunits. Immediately prior to insertion into the plasma membrane, i.e. about 3 h after translation, the alpha'- and beta'-precursor polypeptides were converted to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits. The characteristics of the oligosaccharide moieties of the receptor precursors and products were investigated. The 210-kDa precursor and its two products, the 125-kDa alpha'- and 83-kDa beta'-species, and the mature alpha- and beta-receptor subunits bind tightly to wheat germ lectin, whereas the 190-kDa proreceptor species is not bound. Upon incubation with endoglycosidase H, both the 210- and 190-kDa species are converted to a 180-kDa species. The 125-kDa alpha'- and 83-kDa beta'-species are also cleaved by endoglycosidase H, being reduced in size to 97 and 79 kDa, respectively. Based on their sensitivity to endoglycosidase H and insensitivity to neuraminidase, the oligosaccharide chains of the receptor precursors (190, 210, 125, and 83 kDa) do not contain terminal sialic acid (or other capping sugars). However, near the time of insertion into the plasma membrane, capping of the alpha'- and beta'-species by sialic acid occurs, giving rise to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits, which are partially endoglycosidase H-resistant and neuraminidase-sensitive. When 3T3-L1 adipocytes are treated with tunicamycin, a 180-kDa proreceptor aglycopolypeptide is synthesized which is incapable of undergoing further processing and proteolytic cleavage to the alpha- and beta (or alpha'- and beta'-)-subunits. The 180-kDa species, which appears to be the aglyco-form of hte 190-kDa proreceptor generated by endoglycosidase H, is resistant to trypsin in the intact cell and apparently has not reached the cell surface. Thus, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin re  相似文献   

6.
Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.  相似文献   

7.
We have studied the biosynthesis of rat gastric mucin in stomach segments using an antiserum against rat gastric mucin specific for peptide epitopes. Pulse-chase experiments were performed with [35S]methionine, [3H]galactose, and [35S]sulfate to label mucin precursors in different stages of biosynthesis, which were analyzed after immunoprecipitation. The earliest mucin precursor that could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a 300-kDa protein. The occurrence of N-linked "high-mannose" oligosaccharides on this protein was shown by susceptibility to degradation by endo-beta-N-acetylglucosaminidase H. This precursor could be labeled with [35S]methionine and not with [3H]galactose or [35S]sulfate. The 300-kDa precursor was converted into mature mucin after extensive glycosylation and sulfation. The mature mucin but not the 300-kDa precursor was in part secreted into the medium. Specific inhibition of sulfation with sodium chlorate had no effect on rate and amount of mucin secretion. In addition, we show that two core proteins are expressed in rats, slightly varying in Mr among individual animals.  相似文献   

8.
The insulin proreceptor is a 190-kDa glycoprotein that is processed to mature alpha (135-kDa) and beta (95-kDa) subunits. In order to determine the role of carbohydrate chain processing in insulin receptor biogenesis, we investigated the effect of inhibiting glucose removal from core oligosaccharides of the insulin proreceptor with glucosidase inhibitors, castanospermine and 1-deoxynojirimycin. Cultured IM-9 lymphocytes treated with inhibitors had 50% reduction in surface insulin receptors as demonstrated by ligand binding, affinity cross-linking with 125I-insulin, and lactoperoxidase/Na 125I labeling studies. Degradation rates of surface labeled receptors were similar in both control and inhibitor-treated cells (t1/2 = 5 h); thus, accelerated receptor degradation could not account for this reduction. Biosynthetic labeling experiments with [3H]leucine and [3H]mannose identified an apparently higher molecular size proreceptor (approximately 205 kDa) that failed to show the characteristic decline with time as seen in the normal 190-kDa proreceptor. Along with this finding, the biosynthetic label appearing in the mature subunits was reduced in these inhibitor-treated cells. Endoglycosidase H treatment of both precursors produced identical 170-kDa bands. Carbohydrate chains released from the 205-kDa precursor by endoglycosidase H migrated in the same position as the Glc2-3Man9GlcNAc standards when separated by high performance liquid chromatography, whereas the 190-kDa proreceptor oligosaccharides migrated similar to the Man7-9GlcNAc chains. Although the mature subunits of control and inhibitor-treated cells demonstrated equal electrophoretic mobility, the endoglycosidase H-sensitive oligosaccharides of the mature subunits in treated cells also contained residues that migrated similar to the Glc2-3Man9GlcNAc standards. Thus, glucose removal from core oligosaccharides is apparently not necessary for the cleavage of the insulin proreceptor, but does delay processing of this precursor, which probably accounts for the reduction in cell-surface receptors.  相似文献   

9.
Biosynthesis and processing of the mannose receptor in human macrophages   总被引:4,自引:0,他引:4  
The biosynthesis and processing of the human mannose receptor has been studied in monocyte-derived macrophages. Adherent cells were labeled for 60 min with Trans35S (a mixture of 35S-labeled methionine and cysteine), chased, and subjected to immunoprecipitation by antibody raised against the human placental receptor. The antibody immunoprecipitated a single protein of molecular mass 162 kDa; precipitation of the labeled receptor could be inhibited by placental receptor. The results presented demonstrate that the receptor is synthesized as a 154-kDa precursor which is processed to 162 kDa in 90 min. The precursor is a glycoprotein bearing endoglycosidase H-sensitive oligosaccharides; the 162-kDa form is endoglycosidase H-resistant but peptide:N-glycanase-sensitive. Desialylation of the mannose receptor with neuraminidase generates a protein which is recognized by peanut agglutinin, a lectin that specifically binds desialylated O-linked oligosaccharides. Thus, the human macrophage mannose receptor bears both N- and O-linked oligosaccharide chains. Newly synthesized mannose receptor exhibits a half-life of 33 h as determined by pulse-chase studies. This indicates that on the average, each molecule of receptor recycles between the cell surface and endosomes hundreds of times before degradation.  相似文献   

10.
Biosynthesis and intracellular transport of the mouse macrophage Fc receptor   总被引:11,自引:0,他引:11  
The membrane insertion, processing, and intracellular transport of the mouse macrophage Fc receptor for IgG1/IgG2b was studied using specific mono- and polyclonal anti-receptor antibodies. By immunoprecipitation from Triton X-114 lysates of radiolabeled J774 cells, we determined that the mature 60-kDa receptor is a transmembrane glycoprotein which is synthesized in the rough endoplasmic reticulum as a 53-kDa precursor. Digestion of the precursor with endo-beta-N-acetylglucosaminidase F demonstrated that the receptor consisted of a 37-kDa polypeptide to which four asparagine-linked oligosaccharides were attached. Proteinase K treatment of isolated microsomes indicated that the receptor also has a putative 15-kDa cytoplasmic domain apparently recognized by at least one anti-Fc receptor monoclonal antibody. An additional 15-kDa domain was found to be inaccessible to proteolysis from either side of the membrane. Pulse-chase experiments using [35S]methionine-, [3H]mannose-, and [3H]galactose-labeled cells showed that processing of the receptor's N-linked oligosaccharides occurred rapidly (t1/2 = 15 min) and resulted in the conversion of at least three of the chains to complex endo-beta-N-acetylglucosaminidase H-resistant forms. O-Linked oligosaccharides were not detected. Fc receptor was detected on the plasma membrane 30 min after its synthesis. Transport of newly synthesized receptors to the plasma membrane was slowed but not blocked by incubation of J774 cells at 20 degrees C or by the carboxylic ionophore monensin, although monensin completely inhibited the galactosylation of the receptor.  相似文献   

11.
The human asialoglycoprotein receptor is a 46-kDa membrane glycoprotein. It is initially synthesized as a 40-kDa precursor species possessing two N-linked high-mannose oligosaccharides which is subsequently converted to the 46-kDa mature product upon modification of its oligosaccharides of the complex form [Schwartz, A. L. & Rup, D. (1983) J. Biol. Chem. 258, 11 249-11 255]. To investigate further the biosynthesis of the human asialoglycoprotein receptor, we have utilized a cell-free wheat germ translation system supplemented with dog pancreatic microsomal membranes and programmed with HepG2 and human liver RNA. The primary translation product of the human receptor is a single 34-kDa species and this species is expressed throughout human fetal and adult development. The primary translation product possesses no cleavable signal peptide and is cotranslationally glycosylated to form the 40-kDa precursor species. In addition, the human asialoglycoprotein receptor is co-translationally inserted into microsomal membranes such that a 4-kDa cytoplasmic tail is susceptible to trypsin digestion.  相似文献   

12.
13.
Human complement receptor type 2 (CR2) was biosynthetically labeled by pulsing SB B lymphoblastoid cells for 25 min with [35S]methionine followed by chase in the presence of excess unlabeled methionine. An Mr 134,000 polypeptide represented the major form of the receptor at the end of the pulse period, and within 1 h of chase this disappeared coincident with the appearance of the Mr 145,000 mature form of CR2. Precursor, but not mature, CR2 was sensitive to endoglycosidase H, indicating that maturation of CR2 represented processing of N-linked high mannose oligosaccharides to the complex type. The processing of precursor CR2 was impaired by monensin. In the presence of tunicamycin an Mr 111,000 form of CR2 was synthesized by SB cells, and this did not chase into either precursor or mature CR2. This Mr 111,000 form of CR2 did not incorporate [3H]glucosamine, indicating that it lacked both N- and O-linked oligosaccharide. The half-lives of mature CR2 and nonglycosylated CR2 pulse-labeled in the presence of tunicamycin were 13.8 and 2.8 h, respectively; the turnover rate of B1, a membrane protein normally lacking carbohydrate, was unaffected by the presence of the antibiotic. The percentage of pulse-labeled, nonglycosylated CR2 that was expressed at the cell surface after 1 h of chase in the presence of tunicamycin was 30%, identical to that of mature CR2 in cells chased in the absence of the antibiotic. However, after 6 h of chase there was no additional net accumulation of nonglycosylated CR2 at the plasma membrane, while the proportion of pulse-labeled mature CR2 at this site had risen to 81%. Therefore, N-linked oligosaccharides are essential for the stability of CR2 and have some role in its plasma membrane expression. In contrast, the observation that all three forms of CR2 bound to Sepharose C3 indicates that oligosaccharides are not necessary for the interaction between CR2 and its complement ligand.  相似文献   

14.
The adenosine deaminase-binding protein has previously been localized to the cell surface of human fibroblasts (Andy, R. J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925). In this study we examine the biosynthesis of binding protein in human fibroblasts, human hepatoma HepG2 cells, and a human kidney tumor cell line. Binding protein immunoprecipitated from radioiodinated detergent-extracted fibroblast membranes has a molecular weight of 120,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional band of Mr 100,000 is also present which we believe is a result of proteolysis of the 120,000 band. Purified soluble kidney binding protein has an Mr of 112,000. Binding protein from fibroblasts pulse-labeled with [35S]methionine for 15 min migrates as a 110-kDa band on sodium dodecyl sulfate-polyacrylamide gels. Within 30-60 min of chase, the intensity of the 110-kDa band is diminished, and a 120-kDa band has appeared. Binding protein reaches the cell surface of fibroblasts within 30-60 min of chase. The same results are obtained with the other cell lines studied. Thus, binding protein is initially synthesized as a precursor of 110 kDa which chases into a 120-kDa mature form. The shift of 10 kDa is probably due to processing of its oligosaccharide chains since soluble kidney-binding protein contains 7-9 complex N-linked chains. Upon endoglycosidase H treatment, the 110,000 precursor shifts to a Mr of 89,000 while the 120,000 mature band shifts to 115,000, consistent with the presence of 7-9 high mannose chains on the precursor and 1-2 high mannose chains on the mature form. These results and the presence of complex N-linked chains on binding protein were confirmed by lectin affinity chromatography of glycopeptides derived from [2-3H]mannose-labeled binding protein. Analysis of [6-3H]glucosamine-labeled binding protein indicates the presence of 1 sialic acid residue per chain.  相似文献   

15.
Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line   总被引:3,自引:0,他引:3  
The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.  相似文献   

16.
Tunicamycin, a specific inhibitor of N-glycosylation, was used to study the function of asparagine-linked oligosaccharides of the low density lipoprotein (LDL) receptor in cultured human skin fibroblasts. When cells were preincubated in the presence of 0.5 micrograms/ml of the drug the incorporation of [3H]mannose into the receptor was completely prevented and that of [3H]glucosamine was reduced to approximately 41% of the control value. The [35S]methionine radioactivity detected in receptor core protein of tunicamycin-treated cells was about 52% of that measured in the receptor of control cells. The decrease in the radioactivity was similar in both the mature receptor as well as in its precursor form, and it was significantly greater than that found in total protein. The rates of receptor degradation in control- and tunicamycin-treated cells were comparable. Neither cell surface appearance of the newly synthesized LDL receptor nor its recycling were affected by tunicamycin. However, the LDL receptor produced in tunicamycin-treated cells was smaller in molecular size, and it exhibited an about 50% lower binding capacity when compared with its counterpart synthesized in control cells. This indicates that there is a relationship between N-glycosylation and the ligand binding activity of the LDL receptor. The possible role of asparagine-linked oligosaccharides in optimizing the biological activity of the LDL receptor is discussed.  相似文献   

17.
Abstract: Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic compartments.  相似文献   

18.
The biosynthesis of nonspecific lipid transfer protein (nsLTP) was investigated. Total RNA of rat liver was translated in a rabbit reticulocyte lysate cell-free protein-synthesizing system with [35S]methionine as label. The immunoprecipitation of translation products with affinity-purified anti-nsLTP antibody yielded 14.5- and 60-kDa [35S]polypeptides. The molecular mass of the former polypeptide was approximately 1.5 kDa larger than that of the purified mature nsLTP (13 kDa). The site of synthesis of nsLTP was studied by in vitro translation of free and membrane-bound polyribosomal RNAs followed by immunoprecipitation. mRNA for both the 14.5- and 60-kDa polypeptides were found predominantly in the free polyribosomal fraction in both normal and clofibrate-treated rats. Clofibrate, a hypolipidemic drug that proliferates peroxisomes, did not increase the relative amount of nsLTP mRNA in rat liver. Pulse-chase experiments in rat hepatoma H-35 cells suggested that nsLTP was synthesized as a larger precursor of 14.5 kDa and converted to a mature form of 13 kDa. We have recently shown that nsLTP is highly concentrated in peroxisomes in rat hepatocytes [Tsuneoka et al. (1988) J. Biochem. 104, 560-564]. Taken together, these results suggest that nsLTP is synthesized as a larger precursor of 14.5 kDa on cytoplasmic free polyribosomes, then post-translationally transported to peroxisomes, where the precursor is presumably proteolytically processed to its mature form of 13 kDa. The relationship between the 13-kDa nsLTP and the 60-kDa polypeptide is also discussed.  相似文献   

19.
When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues.  相似文献   

20.
Antibodies to phosphotyrosine were used in immunoprecipitation experiments to determine if post-translational modification of the platelet-derived growth factor (PDGF) receptor was required for the acquisition of ligand-induced tyrosine kinase activity. In intact Balb/c 3T3 fibroblasts, only the fully processed 180-kDa receptor was activated (tyrosine-phosphorylated) by PDGF. In a cell-free assay, however, the tyrosine-phosphorylated forms of the 160- and 145-kDa PDGF receptor precursors were also detected. These activated precursors were immunoprecipitated after brief (5-15 min) metabolic labeling periods. Thus the receptor could bind PDGF and induce tyrosine kinase activity shortly after translation. Unlike the mature form of the receptor, the 160-kDa receptor precursor was resistant to digestion with endo-alpha-N-acetylgalactosaminidase and thus did not contain O-linked oligosaccharides. Since this receptor precursor was activated by PDGF in the cell-free assay, the addition of O-linked sugars must not be necessary for ligand binding activity. Incubation of cells with tunicamycin completely inhibited N-linked glycosylation of the PDGF receptor. Nevertheless, PDGF still induced tyrosine phosphorylation of the 130-kDa aglycoreceptor in lysates of tunicamycin-treated cells. Thus, the addition of N-linked oligosaccharides was also not required for receptor activation. These findings show that the PDGF receptor acquired the ability to be activated by ligand cotranslationally or immediately after translation and that the addition of N- or O-linked oligosaccharides was not required for ligand binding and tyrosine kinase activities.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号