共查询到20条相似文献,搜索用时 62 毫秒
1.
J A Whitsett G Ross T Weaver W Rice C Dion W Hull 《The Journal of biological chemistry》1985,260(28):15273-15279
Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion. 相似文献
2.
Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells 总被引:8,自引:0,他引:8
Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes. 相似文献
3.
Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration. 相似文献
4.
Binding and uptake of pulmonary surfactant protein (SP-A) by pulmonary type II epithelial cells 总被引:4,自引:0,他引:4
R M Ryan R E Morris W R Rice G Ciraolo J A Whitsett 《The journal of histochemistry and cytochemistry》1989,37(4):429-440
A glycoprotein of Mr 26-36,000 (SP-A) is an abundant phospholipid-associated protein in pulmonary surfactant. SP-A enhances phospholipid reuptake and inhibits secretion by Type II epithelial cells in vitro. We have used two electron microscopic cytochemical methods to demonstrate selective binding and uptake of SP-A by rat pulmonary Type II epithelial cells. Using an immunogold bridging technique, we showed that SP-A binding was selective for Type II cell surfaces. Binding was dose dependent and saturable, reaching maximal binding at approximately 10 ng/ml. On warming to 23 degrees C, SP-A binding sites were clustered in coated pits on the cell surface. To characterize the internalization and intracellular routing of SP-A, we used the biotinyl ligand-avidin-gold technique. Biotinyl SP-A was bound by rat Type II epithelial cells as described above. On warming, biotinyl SP-A was seen in association with coated vesicles and was subsequently located in endosomes and multivesicular bodies. Biotinyl SP-A-gold complexes were seen in close approximation to lamellar bodies 10-60 min after warming. Binding of biotinyl SP-A was inhibited by competition with unlabeled SP-A. These results support the concept that Type II epithelial cells bind and internalize SP-A by receptor-mediated endocytosis. This newly described uptake system may play a role in the recycling of surfactant components or mediate the actions of SP-A on surfactant phospholipid secretion. 相似文献
5.
Prothrombin biosynthesis: characterization of processing events in rat liver microsomes 总被引:3,自引:0,他引:3
Plasma and hepatic microsomal forms of rat prothrombin have been compared by sodium dodecyl sulfate-polyacrylamide electrophoresis and isoelectric focusing. The major prothrombin species that accumulated in the microsomes of rats treated with warfarin had a molecular weight of 78 500 and a pI in 8 M urea of 6.3-6.5. Plasma prothrombin had a molecular weight of 83 500 and a pI of 5.3-5.7. Microsomes from normal rat liver contain a second pool of precursor with a molecular weight of 83 500, and digestion with the glycosidase Endo H indicated that this form has been processed to contain complex carbohydrates, while the Mr 78 500 form is a high mannose form and is the substrate for the vitamin K dependent carboxylase. Treatment of rats with tunicamycin revealed that glycosylation was not essential for carboxylation or secretion from the liver. Comparison of the aglyco forms of prothrombin and its precursors suggests that the intracellular forms contain a basic, Mr approximately 1500 peptide that is missing from the plasma form of prothrombin. 相似文献
6.
J E Russell W V Walker R J Fenster D J Simmons 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1985,180(2):375-381
The circadian patterns of bone collagen formation were studied in vitro to ascertain whether the biorhythmic profiles previously measured in vivo reflect true differences in collagen synthesis. Alteration of amino acid pool sizes did not negate the circadian-stage differences in bone collagen production. Evaluations of proline uptake and transport, as well as collagenous protein turnover, demonstrated that the intracellular assembly and secretion of bone collagenous protein during the dark span is truly decreased relative to that during the light period. It was further affirmed that PTH is essential for maintenance of the normal circadian collagen synthesis rhythms. 相似文献
7.
E M Haller S A Shelley M R Montgomery J U Balis 《The journal of histochemistry and cytochemistry》1992,40(10):1491-1500
Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant. 相似文献
8.
Yi Song Guodong Fang Haitao Shen Hui Li Wenbing Yang Bing Pan Guowei Huang Guangyu Lin Lian Ma Belinda Willard Jiang Gu Lemin Zheng Yongyu Wang 《PloS one》2012,7(10)
Surfactant protein A2 (SP-A2) plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA), a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2. 相似文献
9.
The intracellular pathways and the kinetics of metabolism of surfactant apoprotein and lipid, which may be recycled from the alveolar space, are largely unknown. We used a lipid-apoprotein complex made from liposomes of pure lipids in a ratio found in mammalian pulmonary surfactant plus surfactant apoprotein (SP-A, Mr = 26,000-36,000) to test some possible relationships in the recycling of these major surfactant components between intrapulmonary compartments. After intratracheal instillation of 80 microliters of an apoprotein-liposome mixture with separate radiolabels in the lipid and the apoprotein, rats were killed at times from 8 min to 4 h later. The lungs were lavaged with saline, and subcellular fractions were isolated on discontinuous sucrose density gradients. Both the [14C]lipid radiolabel and the 125I-apoprotein radiolabel demonstrated a time-dependent increase in radioactivity recovered in a lamellar body-enriched fraction. Uptake of the radiolabels into other subcellular fractions did not exhibit a clear-cut time dependence; more of the protein than the lipid radiolabel was found in the Golgi-rich and microsomal fractions. We conclude that both the lipid and apoprotein portions of lung surfactant are taken up by lung cells and are incorporated into secretory granules of the cells. 相似文献
10.
Surfactant proteins (SP) have an important impact on the function of the pulmonary surfactant. In contrast to humans, rat
lungs are immature at birth. Alveolarization starts on postnatal day 4. Little is known about the distribution of SP during
postnatal alveolarization. By immunoelectron microscopy, we studied the distribution of SP-A, SP-D, SP-B, and precursors of
SP-C in type II pneumocytes before, near the end and after alveolarization and in mature lungs. We determined the subcellular
volume fractions and the relative labeling index to obtain information about preferential labeling of compartments and non-randomness
of labeling. Independently of alveolarization, the overall cellular distribution of SP was non-random. A preferential labeling
for SP-A and SP-D was found in small vesicles and multivesicular bodies (mvb). SP-B and precursors of SP-C were localized
in mvb and lamellar bodies (lb). There are no postnatal changes in labeling for all three SP in these compartments. Labeling
intensity for SP-B in lb increased in close correlation with a significant increase in the volume fractions of lb during alveolarization.
Our results support the concept that postnatal alveolarization in rat lungs is associated with significant increases in the
SP-B content in lb and volume fraction of lb in type II pneumocytes. The postnatal compartment-specific distribution of SP-A,
precursors of SP-C and SP-D does not change.
Both the authors Andreas Schmiedl and Matthias Ochs contributed equally to this work. 相似文献
11.
Biosynthesis and regulation of rat alpha 1-inhibitor3, a negative acute-phase reactant of the macroglobulin family. 总被引:1,自引:0,他引:1
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T Geiger Y Lamri T A Tran-Thi F Gauthier G Feldmann K Decker P C Heinrich 《The Biochemical journal》1987,245(2):493-500
The biosynthesis of rat alpha 1-inhibitor3, a negative acute-phase reactant specifically found in rodents, was studied in vitro in a cell-free translation system from rabbit reticulocytes, in rat hepatocyte primary cultures and in vivo by immunocytochemistry using normal and turpentine-injected rats. By sucrose-gradient centrifugation and subsequent translation of the fractionated RNA in vitro it was found that the mRNA coding for alpha 1-inhibitor3 exhibited a size of about 28S. For the alpha 1-inhibitor3 translated in vitro an apparent Mr of 155,000 was determined. A continuous decrease in the level of alpha 1-inhibitor3 in serum during experimental inflammation induced by turpentine injection was demonstrated by means of quantitative 'rocket' immunoelectrophoresis. This result agrees with the observation by immunocytochemistry of a drastic decrease in alpha 1-inhibitor3 levels in hepatocytes 24 h after turpentine injection. At that time alpha 1-inhibitor3 is mainly located in the Golgi apparatus, whereas it is also present in the membranes of the rough and smooth endoplasmic reticulum when normal liver is used. All hepatocytes, but no other hepatic cells, contain alpha 1-inhibitor3. When hepatocyte primary cultures were labelled with [35S]methionine and alpha 1-inhibitor3 was immunoprecipitated from the hepatocyte medium and the supernatant of homogenized cells, two different forms of alpha 1-inhibitor3 were found. The intracellular form of alpha 1-inhibitor3, with an apparent Mr of 173,000, is characterized by oligosaccharide side chains of the high-mannose type. The form of alpha 1-inhibitor3 in the medium exhibited an Mr of 186,000 and carried carbohydrate side chains of the complex type. After labelling hepatocytes with radioactive sugars, [3H]mannose was found in both forms of alpha 1-inhibitor3, whereas [3H]fucose and [3H]galactose were incorporated only into the form found in the medium. In the presence of tunicamycin an unglycosylated alpha 1-inhibitor3 with an apparent Mr of 154,000 was found in cells and in the medium. In a pulse-chase experiment it was shown that inhibition of glycosylation by tunicamycin resulted in a marked delay of secretion of alpha 1-inhibitor3. Thus the oligosaccharide side chains of alpha 1-inhibitor3 play an important role during its transport into the medium. 相似文献
12.
Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice 总被引:3,自引:0,他引:3
W F Voorhout T Veenendaal H P Haagsman A J Verkleij L M van Golde H J Geuze 《The journal of histochemistry and cytochemistry》1991,39(10):1331-1336
Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice. 相似文献
13.
To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Sepharose 4B in a Ca(2+)-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylglycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca(2+)-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca(2+)-dependent LB-binding protein identified in the bronchoalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body. 相似文献
14.
Regulation of alpha 1-acid glycoprotein externalization and intracellular accumulation in glucocorticoid-induced rat hepatoma cells 总被引:3,自引:0,他引:3
alpha 1-Acid glycoprotein (alpha 1-AGP) is a glucocorticoid inducible gene product that is synthesized and secreted by certain rat hepatoma tissue culture (HTC) cell lines such as M1.54. Exposure to monensin, a Na+-K+ ionophore, causes a significant redistribution of alpha 1-AGP into two distinct fractions; immunoprecipitation of [35S]methionine-labeled proteins revealed that a 27% decrease in secretion accounts for a sixfold increase in accumulation of a stable intracellular species. The new intracellular alpha 1-AGP is more heterogeneous than normal while the extra-cellular form is 6000 Da smaller than normal. These effects are due to selective alterations in carbohydrate maturation; endo-beta-N-acetylglucosaminidase H (endo H) digestion demonstrated that both alpha 1-AGP species contain variable numbers of endo H-resistant oligosaccharide side chains ranging between zero and five. Ricin affinity chromatography revealed that the attachment of galactose residues is strikingly correlated with alpha 1-AGP externalization while neuraminidase digestions demonstrated that sialic acid attachment appears unessential for its secretion. Taken together, our results suggest that in the presence of monensin the cellular transport of intracellular destined and externalized alpha 1-AGP proceeds in common through the early segments of the Golgi and at a point prior to or at the compartment containing galactosyl transferase, alpha 1-AGP becomes committed for secretion. 相似文献
15.
Evidence for the involvement of an acidic compartment in the processing of myeloperoxidase in human promyelocytic leukemia HL-60 cells 总被引:1,自引:0,他引:1
The observation that myeloperoxidase precursor and larger intermediate (Mr 91,000 and 81,000, respectively) were extracted in the presence of detergent from isolated granule fractions of human promyelocytic leukemia HL-60 cells under mildly acidic conditions was investigated. In contrast, under conditions of neutral pH, only the Mr 74,000 intermediate and mature species were extracted. Extraction of the Mr 91,000 and 81,000 forms was also enhanced in the presence of EDTA. Kinetic studies of the processing of the different myeloperoxidase species confirmed the intermediate nature of the Mr 81,000 and 74,000 forms. Support for a role of an acidic intracellular compartment was obtained through evidence that the acid-extractable precursor and intermediates accumulated in HL-60 cells which had been treated with 1 microM monensin. Under these conditions, the production of mature heavy (Mr 63,000) and light (Mr 13,500) subunits of myeloperoxidase was consistently inhibited by greater than 40% over a 16-h period. The effects of monensin on processing of myeloperoxidase were completely reversed if monensin was removed during this 16-h period. These data support the idea that an acidic compartment may be involved in the transport of myeloperoxidase precursors to azurophil granules and/or their processing to a smaller intermediate form (Mr 74,000) of the enzyme. 相似文献
16.
K Sakai T Kohri Y Kawaguchi M N Kweon Y Kishino 《Cellular and molecular biology, including cyto-enzymology》1992,38(8):853-860
Alveolar macrophages (AM) which are separated into four fractionated subpopulations (I, II, III and IV), represented differential immunohistochemical staining with antibody against pulmonary surfactant protein A (SP-A). In light microscopy, the least dense AM (fraction I) were intensely stained with antibody to SP-A in numerous granules of the cytoplasm, whereas the most dense cells (fraction IV) showed immuno-reactivity with the antibody in several granules distributed in the spreading and elongating cytosol. By Western blot analysis, antibody to SP-A recognized a triplet of nature molecules of SP-A in AM lysate. However, the antigen of the AM lysate almost disappeared when the cells were cultured for more than two days, which indicate that AM do not synthesize SP-A and have digested intracellular SP-A during the cultivation. Immunoelectron microscopically, AM of fraction IV sometimes had very large vacuoles including lamellar body-like structures, probably pulmonary surfactant immediately after taken up from the alveolar lumen by them, which were heavily deposited with gold particles indicating antigenic site of SP-A. Whereas cells of fraction I contained numerous cytoplasmic vacuoles that were frequently labelled with the immuno-gold particles and were not associated with lamellar body-like structures, which may indicate that the materials in the vacuoles are digesting. The results of this experiments suggest that pulmonary surfactant, layered on the alveolar epithelium, is in part taken up by higher dense AM and is digested during a process of their maturation in the direction of lower dense cells, which undergo an important role of metabolism of pulmonary surfactant by AM subpopulations. 相似文献
17.
Swainsonine treatment accelerates intracellular transport and secretion of glycoproteins in human hepatoma cells 总被引:2,自引:0,他引:2
We are interested in determining whether carbohydrates are important regulatory determinants in the intracellular transport and secretion of glycoproteins. In the present study, we have used swainsonine, an indolizidine alkaloid, to modify the structure of N-glycosidically linked complex oligosaccharides. By inhibiting Golgi mannosidase II, swainsonine prevents the trimming of GlcNAc(Man)5(GlcNAc)2 to GlcNAc-(Man)3(GlcNAc)2, resulting in the formation of hybrid-type oligosaccharides. We find, from pulse-chase experiments using [35S]methionine and immunoprecipitation of individual proteins from culture media, that swainsonine treatment (1 microgram/ml) accelerated the secretion of glycoproteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin) by decreasing the lag period by 10-15 min relative to untreated cultures. The enhanced secretion was specific for glycoproteins since the secretion of albumin, a nonglycoprotein, was unaffected. When alpha 1-antitrypsin was immunoprecipitated from the cell lysates, sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorographic analysis demonstrated that the conversion of the high-mannose precursor to the hybrid form in swainsonine-treated cells occurred more rapidly (by about 10 min) than the conversion to the complex form in control cells. Since both the hybrid and complex forms of alpha 1-antitrypsin are terminally sialylated by sialyltransferase in the trans-Golgi, these results suggest that swainsonine-modified glycoproteins traverse the Golgi more rapidly than their normal counterparts. Therefore, accelerated transport within this organelle may account for the decreased lag period of glycoprotein secretion in the swainsonine-treated cultures. 相似文献
18.
K H?rd J B Damm M P Spruijt A A Bergwerff J P Kamerling G W Van Dedem J F Vliegenthart 《European journal of biochemistry》1992,205(2):785-798
The N-linked carbohydrate chains of the beta subunit of human chorionic gonadotropin (hCG-beta) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Subsequently, the O-linked oligosaccharides were split off from the N-deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion-exchange chromatography on Mono Q, HPLC on Lichrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. 1H-NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N-linked oligosaccharides. Residues not written in bold letters are variably present. [formula: see text] The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG-beta (26% vs 2%). The same holds for the alpha 1-6-fucosylation of the asparagine-bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of alpha 2-6-linked Neu5Ac in the most abundant N-glycans, are new features for hCG-beta. The major O-linked carbohydrate chains identified are the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol and the hexasaccharide Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6(Neu5Ac alpha 2-3Gal beta 1-3)GalNAc-ol, both also found in normal urinary hCG. In addition, two novel O-glycans were characterized: [formula: see text] 相似文献
19.
Biosynthesis and processing of the alpha subunit of the voltage-sensitive sodium channel in rat brain neurons 总被引:10,自引:0,他引:10
The sodium channel from rat brain is a complex of alpha (260 kd), beta 1 (36 kd), and beta 2 (33 kd) subunits. The alpha and beta 2 subunits are linked by disulfide bonds. The earliest biosynthetic precursor of the alpha subunit is a 203 kd core polypeptide with sufficient high-mannose carbohydrate chains to increase its apparent size to 224 kd. It is processed to 224 kd and 249 kd precursor forms containing complex carbohydrate chains before it achieves the mature size of 260 kd. Most newly synthesized alpha subunits are not disulfide-linked to beta 2 subunits, but remain as a metabolically stable pool of intracellular subunits. alpha subunits disulfide-linked to beta 2 are found preferentially at the cell surface. A possible role for this intracellular pool as a rate-limiting step in the regulation of the cell surface density and localization of sodium channels in developing neurons is proposed. 相似文献
20.
B R McAuslan W Reilly G N Hannan K Schindhelm B Milthorpe B A Saur 《Experimental cell research》1988,176(2):248-257
The proline analogs cis-4-hydroxy-L-proline, 3,4-dehydro-L-proline, and 2-azetidinecarboxylic acid induced increases in the migration rate of retinal capillary endothelial cells, aortal endothelial cells, corneal endothelial cells, aortal smooth muscle cells, and retinal pericytes. cis-4-hydroxy-D-proline did not. The optimal concentration for migration induction by any of the active agents was approximately 10(-5) M. At higher concentrations (5 x 10(-4) M) migration was not induced or was inhibited. When tested by subcutaneous implant assays in rabbits, cis-4-hydroxy-L-proline and 2-azetidinecarboxylic acid consistently elicited a marked angiogenic response. Whereas these compounds are known to modulate collagen synthesis and secretion, the concentrations at which they are effective inducers of migration suggest that they may have a more specific target than general collagen synthesis. 相似文献