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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DEAE-cellulose chromatography and SDS-PAAG electrophoresis were used to study proteins from endoplasmic reticulum of smooth and rough enterocyte membranes in the vitamin D-replete and deficient rat. The membrane-bound vitamin-D-dependent protein with a molecular weight of 21-25 kDa is shown to consist of the endoplasmic reticulum of smooth membranes. The intensive binding of calcium by these membranes is established.
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PMID:[Vitamin D-dependent protein of smooth endoplasmic reticulum membranes of rat enterocytes]. 318 50

The phenotype of smooth muscle cells (SMCs) in the aortic media of 7 human fetuses (14-20 weeks of gestation) was examined with transmission electron microscopy, immunofluorescence microscopy, and gel electrophoresis of the cytoskeletal and cytocontractile proteins. Ultrastructurally, virtually all medial cells were identified as SMCs having a poorly differentiated phenotype with a cytoplasm rich in rough endoplasmic reticulum and organelles, and with only a few myofilaments. All medial cells stained intensely with antibodies to vimentin, but only in a 20-week-old fetus could we find a few SMCs staining with antibodies to desmin. Nor was desmin detectable with SDS gel electrophoresis followed by immunoblotting, while clear bands corresponding to vimentin, myosin, and actin were present. In isoelectric focusing and two-dimensional gel electrophoresis beta-actin was the most prominent of the 3 actin isoforms in all cases. The present results show that SMCs in the media of fetal human aorta have a poorly differentiated phenotype, which morphologically and biochemically resembles that previously described in the aorta of fetal and newborn rat, in the arterial intima after endothelial injury, in atherosclerotic lesions, and after spontaneous modulation of medial SMCs in culture.
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PMID:Characterization of the phenotype of smooth muscle cells in human fetal aorta on the basis of ultrastructure, immunofluorescence, and the composition of cytoskeletal and cytocontractile proteins. 321 79

Transport of Ca2+ by the ATP-dependent Ca2+ pump has been demonstrated previously in rat intestinal basolateral-membrane vesicles. To identify the Ca2+-pump protein, duodenal basolateral membranes were phosphorylated with [gamma-32P]ATP in the presence of Ca2+ and La3+, under conditions conducive for maximal formation of the phosphorylated intermediate of the Ca2+ pump. Four major phosphoprotein bands were seen on autoradiograms of acidic SDS/polyacrylamide gels; the properties of a phosphoprotein (pp) at 130 kDa (pp130) were consistent with those expected for the plasma-membrane Ca2+ pump. This phosphoprotein was markedly enhanced by La3+, exhibited the characteristics of an acyl-phosphate bond, was preferentially phosphorylated from ATP and inhibited by micromolar concentrations of vanadate. Another phosphoprotein of 115 kDa possibly represented the endoplasmic reticulum Ca2+ pump or a fragment of pp130. Other phosphoproteins of 75 and 95 kDa were predominantly expressions of alkaline phosphatase. Formation of pp130 was highest in duodenal basolateral-membrane preparations when compared with those of jejunum and ileum or other subcellular fractions. A similar correlation between Ca2+-pump activity and pp130 formation was not found in membranes from villus-tip and crypt cells or in vitamin D-deficient animals. pp130 was isolated as a single phosphoprotein by calmodulin-affinity chromatography. We conclude that pp130 represents the phosphorylated intermediate of the rat intestinal basolateral-membrane Ca2+ pump, which can be separated from other phosphoproteins using its properties as a calmodulin-binding protein.
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PMID:Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes. 322 32

A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich 'shells' followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (X 10(3] Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (X 10(3] Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (X 10(3] Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies. Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+. A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55 X 10(3) Mr protein, respectively, with endoplasmin and the 55 X 10(3) Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity. Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55. These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.
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PMID:Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum. 325 4

A temperature-sensitive mutant of Chinese hamster fibroblasts with a defect in glycoprotein synthesis is investigated after transfection and amplification of the gene for the human EGF receptor. We demonstrate that at the nonpermissive temperature a partially glycosylated species of the receptor accumulates in the endoplasmic reticulum. The oligosaccharides present are the high mannose types, since they can be removed completely by treatment with endoglycosidase H. Pulse-chase experiments show that the abnormal species of the receptor cannot be chased to a form that is either resistant to endoglycosidase H, or altered in its mobility on SDS polyacrylamide gels. The abnormal species of the receptor appears within the first hour of a shift to the nonpermissive temperature, and no further changes are observed upon prolonged incubation of cells at 40 degrees C. However, after 3-4 hours immunoprecipitations of the receptor yield another protein, which has properties very similar, if not identical, to the glucose-regulated protein GRP78. The induction of this protein at 40 degrees C can be suppressed completely with an inhibitor of RNA synthesis, without any effect on the glycosylation defect, or on the accumulation of the EGF receptor in the endoplasmic reticulum.
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PMID:Temperature-sensitive Chinese hamster cell mutant with a defect in glycoprotein synthesis: accumulation of the EGF receptor in the endoplasmic reticulum and the role of the glucose-regulated protein GRP78. 329 37

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.
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PMID:Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum. 343 65

To determine the subcellular sites of sulfation of thyrotropin (TSH) and free alpha-subunits, mouse thyrotropic tumor minces were incubated simultaneously with [3H]Met and [35S]SO4 for 1 or 3h, homogenized, and fractionated by discontinuous sucrose gradient ultracentrifugation. Dual-labeled TSH or free alpha-subunits were immunoprecipitated, and analyzed by SDS-gel electrophoresis. Endoglycosidase F released all [35S], but little [3H], from the dual-labeled species, indicating that [35S]SO4 was incorporated into oligosaccharides of TSH and free alpha-subunits. Both [35S]TSH and [35S] free alpha-subunits were predominantly in Golgi fractions at 1 and 3 h, but small amounts were also detected in fractions enriched in rough endoplasmic reticulum (RER). Similar distributions of [35S]SO4-labeled species were noted in cell fractions prepared from mouse pituitaries. Pituitaries from hypothyroid mice were incubated with [3H]Met and [35S]SO4 for 2 h, then chased for 4 or 16 h in the absence or presence of 2 uM monensin (Mon) or 10 uM carboxyl cyanide m-chlorophenylhydrazone (CCCP). At 4h, release into the medium of [3H]TSH was inhibited 59% and 86% by Mon and CCCP, respectively; release of [35S]TSH was inhibited 28% and 46%. At 4h, release of [3H]free alpha-subunits was inhibited 58% and 81% by these drugs, respectively; release of [35S]free alpha-subunits was inhibited 6% and 50%. Thus, Mon and CCCP inhibited the release of each [3H] species more than the [35S] species, indicating that most sulfation occurred in Golgi.
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PMID:The subcellular sites of sulfation of mouse thyrotropin and free alpha subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins. 344 83

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride. 351 9

Early in primary culture, arterial smooth-muscle cells undergo a transition from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and of contractility. At the same time, an extensive rough endoplasmic reticulum and a large Golgi complex are formed, and active synthesis of DNA, RNA and proteins commences. In the present study, chemical and immunocytochemical methods were used to investigate the production of extracellular-matrix proteins in relation to this change in phenotypic properties. The results showed that the phase of rapid cellular proliferation that follows the structural modulation of smooth-muscle cells is associated with high rates of collagen and elastin synthesis, as measured by the incorporation of 3H-proline into 3H-hydroxyproline and 3H-valylproline, respectively. SDS-polyacrylamide gel electrophoresis and fluorography indicated that type-I collagen is the main collagen species synthesized by these cells. Smaller amounts of type-V collagen and (although not definitively identified) type-III collagen were also detected. Indirect immunofluorescence and immunoelectron microscopy demonstrated that smooth-muscle cells surround themselves with an incomplete basement membrane, containing laminin and type-IV collagen, and thin fibrils of type-I collagen. Adjacent to these fibrils, aggregates of amorphous, elastin-like material were also found. Our observations confirm and extend earlier notions of a close similarity between the behaviour of arterial smooth-muscle cells during in vitro cultivation and during the early stages of the formation of atherosclerotic lesions.
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PMID:Phenotype modulation in primary cultures of smooth-muscle cells from rat aorta. Synthesis of collagen and elastin. 353 83

In infected BHK21 cells, the glycoproteins G1 and G2 of a temperature-sensitive mutant (ts12) of Uukuniemi virus (UUK) accumulate at 39 degrees C in the Golgi complex (GC) causing an expansion and vacuolization of this organelle. We have studied whether such an altered Golgi complex can carry out the glycosylation and transport to the plasma membrane (PM) of the Semliki Forest virus (SFV) glycoproteins in double-infected cells. Double-immunofluorescence staining showed that approximately 90% of the cells became infected with both viruses. Almost the same final yield of infectious SFV was obtained from double-infected cells as from cells infected with SFV alone. The rate of transport from the endoplasmic reticulum (ER) via the GC to the plasma membrane of the SFV glycoproteins was analysed by immunofluorescence, surface radioimmunoassay and pulse-chase labeling followed by immunoprecipitation, endoglycosidase H digestion and SDS-PAGE. The results showed that: the SFV glycoproteins were readily transported to the cell surface in double-infected cells, whereas the UUK glycoproteins were retained in the GC; the transport to the PM was retarded by approximately 20 min, due to a delay between the ER and the central Golgi; E1 of SFV appeared at the PM in a sialylated form. These results indicate that the morphologically altered GC had retained its functional integrity to glycosylate and transport plasma membrane glycoproteins.
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PMID:Efficient transport of Semliki Forest virus glycoproteins through a Golgi complex morphologically altered by Uukuniemi virus glycoproteins. 354 12


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