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1.
Expression of a developmentally regulated antigen on the surface of skeletal and cardiac muscle cells 总被引:6,自引:4,他引:2
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H36 is a species-specific, cell-surface antigen on differentiating newborn rat skeletal myoblasts and myogenic lines. This membrane antigen has been defined by a monoclonal antibody raised by the fusion of SP 2/0-Ag14 myeloma cells with spleen cells from mice immunized with myotubes derived from the myogenic E63 line. H36 antigen, isolated by immunoaffinity chromatography, is comprised of two polypeptides with apparent molecular weights of 98,000 and 117,000. Fluorescence photometry and radioimmunoassays have been used to follow quantitative and topographic changes in the H36 determinant during myogenesis. H36 is present at a basal level on replicating myoblasts; it increases on prefusion myoblasts and persists on myotubes. At or near the time of prefusion, it becomes concentrated between adjacent aligned myoblasts and localized on membrane "blebs". H36 is present on both skeletal and cardiac cells but absent from a variety of cells that include fibroblasts, neuronal cells, and smooth muscle. There are approximately 4 x 10(5) determinants per myoblast, and the Ka of the antibody is 3.8 x 10(8) liters/mol. The distributions of H36 on the top and attached surfaces of myoblasts and myotubes are distinct, which suggests localized specialization of these surfaces. H36 is an integral membrane component and upon cross-linking, it associates with the detergent-insoluble cytoskeletal framework. Inhibition of myogenesis by 5-bromodeoxyuridine or by calcium deprivation prevents the developmentally associated changes in the expression of H36. H36 is also absent or markedly reduced on the fu- and Ama102 developmentally defective mutant myoblast lines. We conclude that H36 is a muscle-specific, developmentally regulated cell-surface antigen that may have a role in myoblast differentiation and that can be used to determine the embryonic lineages of skeletal and cardiac muscle. 相似文献
2.
Receptor-mediated endocytosis of alpha 2-macroglobulin and transferrin in rat caput epididymal epithelial cells in vitro 总被引:1,自引:0,他引:1
The endocytic activity of epithelial cells from the rat epididymis in vitro has been examined by following the uptake of tracer compounds conjugated to proteins. Transferrin-gold and alpha 2-macroglobulin-gold were taken up initially in coated pits, internalized and sequestered into tubular-vesicular structures, multivesicular bodies and, in the case of alpha 2-macroglobulin, into lysosomes. Uptake could be prevented by an excess of unlabeled protein. Studies using 125I-alpha 2-macroglobulin and 125I-transferrin also showed that the uptake of these proteins was specific and could be displaced with increasing amounts of unlabeled protein. In addition, binding of 125I-transferrin to cells was saturable at 4 degrees C. These studies indicate that transferrin and alpha 2-macroglobulin are taken up by receptor-mediated endocytosis. In contrast, a fluid phase marker, bovine serum albumin-gold (BSA-gold), was initially taken up predominantly in uncoated caveolae rather than coated pits, and could not be displaced with excess BSA. By virtue of their charge, polycationized ferritin and unlabeled colloidal gold were taken up and internalized by adsorptive endocytosis, a pathway which is similar to fluid phase endocytosis. The uptake and internalization of alpha 2-macroglobulin and transferrin differed in a number of respects. Uptake and internalization of alpha 2-macroglobulin but not of transferrin was dependent on extracellular calcium. Only alpha 2-macroglobulin was transferred into lysosomes, whereas transferrin was recycled to the cell surface. Although the proton ionophore, monensin, and the transglutaminase inhibitor, dansylcadaverine, did not stop uptake and internalization of either alpha 2-macroglobulin or transferrin, they did prevent the transfer of alpha 2-macroglobulin to lysosomes. 相似文献
3.
During the differentiation of skeletal muscle, mononucleate myoblasts proliferate, then stop replicating, spontaneously fuse, and express a large number of genes which encode the muscle phenotype. We have used monoclonal antibodies specific for 5-bromodeoxyuridine, myoactin, and equine alpha 2-macroglobulin to follow and establish the sequence of events that surround the transition from a replicating to a differentiating population. Triple-label immunofluorescence microscopy was used to visualize the changes in DNA synthesis, formation of myoactin fibers, and the cessation of endocytosis of alpha 2-macroglobulin that accompany myogenesis. Our results indicate that myoblasts cease actively endocytosing alpha 2-macroglobulin after stopping DNA synthesis but prior to fusion. Formation of myoactin fibers rarely occurs in mononucleate myoblasts and only in post-mitotic cells, but they are common in multinucleate myotubes. We suggest that the regulation of DNA synthesis is critical to normal myogenesis and that detection of incorporated BrdUrd by immunofluorescence, in conjunction with other antibodies and nucleic acid probes, is a convenient method with which to study and sequence the molecular events in single cells as they relate to the transition in DNA synthesis that accompanies differentiation. 相似文献
4.
M C van Dijk J K Kruijt W Boers C Linthorst T J van Berkel 《The Journal of biological chemistry》1992,267(25):17732-17737
The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules. 相似文献
5.
H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis [published erratum appears in J Cell Biol 1992 Jul;118(1):213]
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H36 is a 120,000-D membrane glycoprotein that is expressed during the differentiation of skeletal muscle. H36 cDNA clones were isolated from a lambda UniZapXR rat myotube cDNA library and sequenced. The deduced amino acid sequence demonstrates that H36 is a novel integrin alpha chain that shares extensive homology with other alpha integrins that includes: (a) the GFFKR sequence found in all alpha integrins; (b) a single membrane spanning region; (c) conservation of 18 of 22 cysteines; and (d) a protease cleavage site found in the non-I region integrin alpha chains. The cytoplasmic domain of H36 is unique and additional regions of nonhomology further indicate H36 is distinct from all other alpha chains. In keeping with current nomenclature we designate this alpha chain alpha 7. Northern blots demonstrate that expression of H36-alpha 7 mRNA is regulated both early in the development of the myogenic lineage and later, during terminal differentiation. Detection of H36-alpha 7 mRNA coincides with conversion of H36- myogenic precursor cells to H36+ cells. H36-alpha 7 mRNA is present in replicating myoblasts: expression increases upon terminal differentiation and is markedly reduced in developmentally defective myoblasts. In addition, H36-alpha 7 mRNA is not detected in C3H10T1/2 cells. It is in myotubes derived from myoblasts obtained by treatment of 10T1/2 cells with azacytidine or transfection with MRF4. Immunoblots and immunofluorescence demonstrate that the H36-alpha 7 chain is associated with integrin beta 1. Affinity chromatography demonstrates that H36-alpha 7 beta 1 selectively binds to laminin. The expression of H36-alpha 7 on secondary myoblasts during the development of the limb in vivo corresponds with the appearance of laminin in the limb, with the responsiveness of secondary myoblast proliferation to laminin, and with the onset of increased muscle mass, suggesting that H36-alpha 7 modulates this stage in limb development. We conclude that H36-alpha 7 is a novel alpha integrin laminin binding protein whose expression is developmentally regulated during skeletal myogenesis. 相似文献
6.
Uptake and degradation of insulin and alpha 2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways 总被引:3,自引:0,他引:3
The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin. 相似文献
7.
39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. 总被引:41,自引:0,他引:41
J Herz J L Goldstein D K Strickland Y K Ho M S Brown 《The Journal of biological chemistry》1991,266(31):21232-21238
A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues. 相似文献
8.
Remodeling of the myoblast membrane accompanies development 总被引:4,自引:0,他引:4
We have prepared a library of cloned hybridomas that produce monoclonal antibodies reactive with the surface of E63 rat myoblasts. Using immunofluorescence analysis of antigens on single cells we have studied the expression of determinants at distinct stages of development. Conditions were established for quantitative photometry and were used to confirm the diversity in stage-specific expression that accompanies development. The remodeling of the myoblast membrane also involves stage-specific and transient changes in topography and aggregation of many antigens, and the period surrounding fusion is one of particular activity. The localization of antigens on the upper and attached surfaces of myogenic cells was often distinct, and quantitative and spatial stage-specific reorganizations of antigens differed with respect to these two surfaces. This polarity represents an additional level of complexity in the continuous remodeling of the muscle cell membrane. Comparisons of quantitative and topographic analyses of antigens on E63 cells with Rat-1 fibroblasts and developmentally defective (fu-) myoblasts indicate that the outer membranes of these nonmyogenic cells are distinct from differentiating myoblasts. One determinant, H36, is absent on Rat-1 cells and on all fu- lines tested, and undergoes interesting stage-specific changes in expression and topography. 相似文献
9.
Radioimmunoassay of rat acute-phase alpha 2-macroglobulin 总被引:1,自引:0,他引:1
A double-antibody radioimmunoassay (RIA) to acute-phase alpha 2-macroglobulin was developed for the quantitation of this large macromolecule in physiological fluids. The primary receptor for the RIA was a monospecific antiserum to purified acute-phase alpha 2-macroglobulin which produced a high titre (7.5 . 10(6)) antibody with a strong affinity for rat acute-phase alpha 2-macroglobulin (Ka = 1.24 . 10(11)) as measured by Scatchard analysis. The validity of the assay was confirmed by specificity for rat alpha 2-macroglobulin measured in various physiological fluids as assessed by parallel dose-response curves; and accuracy, measured by the analytical recovery of alpha 2-macroglobulin by the RIA in serum (104 +/- 7%) and buffer (103 +/- 7%), and the correlation (R = 0.999) of measurements of acute-phase alpha 2-macroglobulin-containing samples measured in serum and buffer. Reference acute-phase serum measured by this RIA and by rocket immunoelectrophoresis were 98.6% in agreement. Radioimmunoassay sensitivity was estimated at less than 1.0 ng alpha 2-macroglobulin/ml, measured over a range of 0-160 ng. Precision was assessed by intraassay (2.99 +/- 0.97%) and interassay (8.76 +/- 2.64%) variation. Evaluation confirmed that quantitation of rat acute-phase alpha 2-macroglobulin by this RIA met the criteria of sensitivity, validity and precision. 相似文献
10.
Complexes of labelled proteinases (subtilopeptidase A, trypsin) with serum alpha 1-macroglobulin or alpha 2-macroglobulin are rapidly taken up in vitro by rabbit alveolar macrophages and peritoneal macrophages but not by mixed rabbit peripheral blood leukocytes. Enzyme, not bound to alpha 1- or alpha 2-macroglobulin, does not become associated with alveolar macrophages. Chemically inactivated subtilopeptidase A does not bind to alpha 1- or alpha 2-macroglobulin; chemically inactivated subtilopeptidase A in mixtures with alpha 1 - or alpha 2-microglobulin, does not interact with alveolar macrophages. Blocking experiments confirmed that the interaction of proteinase with alveolar macrophages is complex specific; uptake of labelled complex was prevented by the simultaneous addition of macroglobulin complexes formed with non-labelled subtilopeptidase A, subtilopeptidase B, trypsin or chymotrypsin but not by macroglobulin alone. The findings demonstrate a complex-specific interaction between proteinase-alpha-macroglobulin complexes and macrophages. 相似文献
11.
K Hanaoka T J Pritchett S Takasaki N Kochibe S Sabesan J C Paulson A Kobata 《The Journal of biological chemistry》1989,264(17):9842-9849
To investigate the molecular basis of the differential ability of human, equine, and guinea pig alpha 2-macroglobulins to inhibit hemagglutination and infectivity of a human influenza virus, A/Memphis/102/72 (H3N2), the structures of oligosaccharides released from the three glycoproteins by hydrazinolysis were analyzed comparatively. Approximately seven to eight sugar chains were released from each subunit of two potent inhibitors (equine and guinea pig alpha 2-macroglobulins) and a weak inhibitor (human alpha 2-macroglobulin). More than 70% of the oligosaccharides contained sialic acids in all three cases. Structural analysis of these sialo-oligosaccharides revealed that all of the three glycoproteins contain biantennary oligosaccharides with one and two sialic acids as major sugar chains (70-80% of total sugar chains). Four percent of the biantennary oligosaccharides from equine sample, 10% of those from guinea pig, and 24% of those from human contain a fucosylated trimannosyl core. No triantennary oligosaccharide was detected in equine alpha 2-macroglobulin. However, human and guinea pig alpha 2-macroglobulins contain both fucosylated and nonfucosylated triantennary oligosaccharides. All sialic acid residues occur as the Sia alpha 2----6Gal group. The one unique feature of the carbohydrate groups of equine and guinea pig alpha 2-macroglobulins was the presence of 4-O-Ac-Neu5Ac as 30-50% of the total sialic acids, while human alpha 2-macroglobulin contained only Neu 5Ac. However, 4-O-Ac-Neu5Ac is not responsible for the potent inhibition of influenza virus infection and hemagglutination as will be described in the accompanying paper. 相似文献
12.
Enzyme activities of purine catabolism and salvage, the concentrations of high-energy phosphates and the reutilisation of purine bases and purine nucleosides were studied in rat heart myoblasts and myocytes. Rat heart myoblasts H9c2(2-1) were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum. Reduction of fetal calf serum to 2% for 1 week resulted in a differentiation into myocytes with respect to their morphological features and their enzyme pattern. In differentiated myocytes, activity of 5'-nucleotidase was increased more than 2-fold, and AMP deaminase and creatine kinase activities were more than 10-fold elevated. The concentration of creatine phosphate in differentiated myocytes was doubled compared to that in myoblasts. The uptake into myoblasts and myocytes and the incorporation into adenine nucleotides was highest using adenosine, inosine and adenine uptake rates were intermediate, and hypoxanthine was utilised least. Differentiation of myoblasts into myocytes resulted in a slightly lower overall uptake of adenosine and adenine, whereas about 40% more inosine and hypoxanthine were utilised by myocytes. Increasing the phosphate concentration in the incubation medium up to 50 mmol/l resulted in a stimulation of uptake of all purine compounds tested. This stimulation was more pronounced in myoblasts. 相似文献
13.
Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins 总被引:2,自引:0,他引:2
V Gross P C Heinrich D vom Berg K Steube T Andus T A Tran-Thi K Decker W Gerok 《European journal of biochemistry》1988,173(3):653-659
The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms. 相似文献
14.
Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay. 相似文献
15.
B Dahlmann L Kuehn P C Heinrich H Kirschke B Wiederanders 《Biochimica et biophysica acta》1989,991(2):253-262
From rat skeletal muscle tissue we have isolated and purified a proteolytic activity of molecular mass 750 kDa. The enzyme, designated 'proteinase I', which has been found to be located in capillaries of skeletal muscle tissue, catalyzes the hydrolysis of Z-Phe-Arg-MCA and [14C]methylcasein and this process is activated about 2-fold by ATP. As judged by SDS-polyacrylamide gel electrophoresis the subunit pattern of 'proteinase I' is similar to alpha-macroglobulin. Immunoelectrophoretic analyses of 'proteinase I' with antisera to rat alpha 1-macroglobulin, alpha 2-macroglobulin, and rat liver cathepsins reveal that this high-molecular-mass proteinase is a complex of alpha 1-macroglobulin and the cysteine proteinases cathepsin B, H and L. A similar 'proteinase' has been isolated from rat serum. Two ATP-activated high molecular-mass proteinases that have been previously identified in liver and heart muscle by other investigators equally show a positive immunological reaction with the antiserum raised against 'proteinase I'. From these data, together with results presented in an accompanying paper (Kuehn, L., Dahlmann, B., Gauthier, F. and Neubauer, H.-P. (1989) Biochim. Biophys. Acta 991, 263), we conclude that the ATP-stimulated high-molecular-mass proteolytic activity is partly due to the presence of a complex of alpha-macroglobulin and cysteine proteinases. 相似文献
16.
The biosynthesis and secretion of alpha 2-macroglobulin was studied in rat hepatocyte primary cultures. After immunoprecipitation of alpha 2-macroglobulin from a cell homogenate and the hepatocyte medium, two forms of alpha 2-macroglobulin with app. Mr of 176000 and 182000, respectively, were identified. A precursor-product relationship for the two alpha 2-macroglobulin forms was demonstrated by a pulse-chase experiment. The cellular form of alpha 2-macroglobulin could be deglycosylated by endoglucosaminidase H, whereas the medium form of alpha 2-macroglobulin remained unaffected. On the other hand, only the medium form of alpha 2-macroglobulin was found to be susceptible to neuraminidase. In vitro translation of rat liver poly(A)+ RNA resulted in a translation product of an app. Mr of 162000. 相似文献
17.
K Steube V Gross D H?ussinger T A Tran-Thi K Decker W Gerok P C Heinrich 《Biochemical and biophysical research communications》1986,141(3):949-955
The clearance of the rat acute-phase proteins alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein with no, high-mannose, hybrid or complex type oligosaccharide side chains was determined in the isolated perfused rat liver. The differently glycosylated forms of the three proteins were obtained from rat hepatocyte primary cultures treated with different inhibitors of glycosylation. The complex type forms of the three proteins were essentially not cleared by the liver during 2 h of perfusion. Unglycosylated alpha 2-macroglobulin and alpha 1-acid glycoprotein decreased in the perfusate by about 50% after 2 h; unglycosylated alpha 1-proteinase inhibitor was not taken up by the liver. The high-mannose type forms of the three proteins were nearly totally cleared. After 2 h of perfusion 10%, 45% and 30% of the hybrid type forms of alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, respectively, were cleared. The clearance rates of high-mannose and of hybrid type glycoproteins could be reduced to the rates of complex type glycoproteins by the addition of mannan to the perfusate. It is concluded that complex type glycosylation prevents the uptake of plasma glycoproteins by the liver. 相似文献
18.
T Andus G Ramadori P C Heinrich T Knittel K H Meyer zum Büschenfelde 《European journal of biochemistry》1987,168(3):641-646
Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize alpha 2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized alpha 2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. alpha 2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of the cells, increasing amounts of alpha 2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled alpha 2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. alpha 2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled alpha 2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with alpha 2-macroglobulin. alpha 2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where alpha 2-macroglobulin was synthesized in Ito cells, no synthesis of alpha 1-macroglobulin, alpha 1-inhibitor 3, alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, alpha 1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that alpha 2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases. 相似文献
19.
Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation 总被引:2,自引:0,他引:2
Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h. 相似文献
20.
The type B-specific lectin from the mushroom Marasmius oreades was immobilized onto Sepharose 4B. The immobilized lectin bound murine laminin and bovine thyroglobulin, glycoproteins that contain the Galalpha1,3Galbeta1,4GlcNAc epitope. This epitope is responsible for hyperacute rejection of xenotransplants from lower mammals to humans, Old World monkeys, or apes. The immobilized lectin also bound a fraction of serum proteins from type B human serum but little or none from type A or O(H) serum. The major protein bound from human B serum was a portion of the alpha2-macroglobulin. Treatment of this fraction with N-glycosidase F resulted in decreased molecular weight of bands associated with alpha2-macroglobulin and loss of their M. oreades lectin reactivity, whereas on treatment with coffee bean alpha-galactosidase, this bound fraction also lost reactivity with M. oreades lectin but became reactive with Ulex europaeus I lectin, suggesting the presence of L-fucosyl-alpha1,2-terminated structures. The presence of blood group epitopes on alpha2-macroglobulin has been detected previously by immunological methods, but this is the first isolation and characterization of the specifically glycosylated fraction of this serum protein. The immobilized lectin also bound a number of proteins from pig, rabbit, and rat serum that were distinct in electrophoretic mobility from the human B-serum components and presumably contain the xenotransplantation epitope among their glycan structures. This report further demonstrates the utility of immobilized lectins in isolating and characterizing glycan structures of naturally occurring glycoproteins. 相似文献