UNIPROT:O95477 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Target Concepts:

Query: UNIPROT:O95477 (membrane-bound)
29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship between apoB-100 and the membrane of the endoplasmic reticulum (ER) has been studied by a combination of pulse-chase methodology and subcellular fractionation. HepG2 cells were pulse-labeled with [35S]methionine for 3 min and chased with cold methionine for periods between 0 and 20 min. ApoB-100 and albumin, present in the membrane as well as in the luminal content of the ER vesicles, were isolated after each chase period. The results indicated that apoB-100 was cotranslationally bound to the membrane of the ER, and from this membrane-bound form, was transferred to the lumen after a delay of 10-15 min. Albumin was, as could be expected for a typical secretory protein, cotranslationally sequestered in the lumen of the ER. Apo-B-100-containing lipoproteins present in the microsomal lumen were analyzed by ultracentrifugation in a sucrose gradient. ApoB-100 occurred on rounded particles in three density regions: (i) d 1.1065-1.170 g/ml (Fraction I), (ii) d 1.011-1.045 g/ml (Fraction II), and (iii) d less than 1.011 g/ml (Fraction III). Fraction I, isolated from cells cultured in the absence of oleic acid, contained a homogenous population of particles with a mean diameter of approximately 200 A. Fraction I isolated from cells cultured in the presence of oleic acid was slightly more heterogeneous and had a mean diameter of approximately 250 A. Fractions II and III had mean diameters of 300 and 500 A, respectively. Cholesterol esters and triacylglycerol were the quantitatively dominating lipid constituents of all three fractions. Pulse-chase experiments indicated that Fraction I contained the newly assembled lipoproteins. With increasing chase time, the apoB-100 radioactivity was redistributed from Fraction I to Fractions II and III, indicating that Fraction I is converted into Fractions II and III during the intracellular transfer. Particles corresponding to Fractions II and III were by far the most abundant lipoproteins found in the medium. The results presented support the possibility of a sequential assembly of apoB-100-containing lipoproteins.
...
PMID:Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells. 334 54

The phorbol 12-myristate 13-acetate (PMA)-dependent down-regulation of immunoprecipitable protein kinase C was studied in human breast cancer cell lines that display different growth inhibitions toward the tumor promoter. PMA induces translocation of [35S]methionine-prelabeled cytosolic protein kinase C to membranes, followed by complete degradation of the enzyme (t1/2, 2 hr). PMA does not affect the protein kinase C synthesis; 20-80% of total protein kinase C of control cells was still immunoprecipitable as membrane-bound 74- and 80-kDa protein kinase C-related polypeptides if cells were allowed to incorporate [35S]methionine during PMA exposure for greater than 6 hr. These two proteins lack protein kinase activity and phorbol ester binding but reveal V8 peptide patterns identical to the active forms of protein kinase C (77/80 kDa) of PMA-untreated cells. The amounts of the immunoprecipitable membrane-bound 80-kDa protein kinase C-related polypeptide synthesized during the prolonged PMA treatment appear to inversely correlate with the extent of PMA-mediated growth inhibition of the respective human breast cancer cell line. These data suggest that after homologous down-regulation, functional protein kinase C (77/80 kDa) is replaced by a population of membrane-associated but enzymatically inactive protein kinase C-related polypeptides (74/80 kDa).
...
PMID:Continuous synthesis of two protein-kinase-C-related proteins after down-regulation by phorbol esters. 335 68

The aim of this study was to analyze whether a monoclonal antibody to human milk fat globule membrane-associated antigens, recognized specifically and homogeneously by human breast carcinoma cells but also by normal epithelial cells active in secretion, could be used to restrict the access of antitumoral drugs to cells exposing the epitope. The drug-antibody conjugate to be used is constructed by means of a covalent peptidic linkage stable in extracellular medium but hydrolyzed by lysomal enzymes after endocytosis of the drug-carrier conjugate. This monoclonal antibody specifically immunoprecipitates radioactive material from MCF-7 cells biosynthetically radiolabeled with galactose, glucosamine, palmitic acid, or acetic acid but not with mannose, leucine, or methionine. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, the label migrates as two bands with apparent molecular weights of about 350,000 and 400,000. These bands disappear, or their molecular weight is affected, after treatment of the cells with cycloheximide or of cell lysates with trypsin, Pronase, or neuraminidase but not treatment of the immunoprecipitate with endoglycosidase F. This suggests that these antigens are glycoproteins with O-linked oligosaccharides containing sialic acid in the epitope. By analogy, they should be similar, if not identical, to those recognized by the monoclonal antibodies designated HMFG1 (H. Burchell, H. Durbin, and J. Taylor-Papadimitriou, J. Immunol., 131:508-513, 1983) and DF3 (H. Sekine, T. Ohno, and D.W. Kufe, J. Immunol., 135:3610-3615, 1985). Binding at 4 degrees C of the 3H-labeled antibody by MCF-7 cells indicates the specific attachment of about 1.2 X 10(6) IgG molecules per cells with a Kd of about 14 nM. At 37 degrees C, cells take up the 3H-labeled antibody in amounts much higher than the binding capacity. In addition to cell-associated material, labeled digestion products are released into the culture medium. Cell fractionation by differential centrifugation and isopycnic equilibration on sucrose gradient indicates that the bulk of cell-associated antibody is distributed like the marker enzyme of lysosomes. Although the total uptake of the antibody by the cells is unaffected by either 50 microM chloroquine or 3 micrograms/ml cycloheximide, the release of digestion products is completely inhibited by chloroquine. Antigen-antibody dissociation is pH dependent, since, respectively, 50 and 84% of membrane-bound antibody are released during washing at pH 4.6 and 4.1.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Binding and endocytosis of a monoclonal antibody to a high molecular weight human milk fat globule membrane-associated antigen by cultured MCF-7 breast carcinoma cells. 336 2

The biosynthesis and maturation of human sucrase-isomaltase (SI, EC 3.2.1.48-10), was studied in cultured small intestinal biopsy specimens and mucosa explants. Pulse-chase experiments with [35S]methionine revealed one high mannose intermediate of Mr = 210,000 (pro-SIh) which was processed at a slow rate to an endo H-resistant, mature form of Mr = 245,000 (pro-SIc). The fully core-glycosylated form (Mr = 212,000) was detected only when 1-deoxynojirimycin was added to the culture medium, thus indicating that the core sugars undergo rapid processing by rough endoplasmic reticulum membrane-bound glycosidases. The data presented showed that trypsin specifically and instantaneously (within 1 min) cleaves pro-SIc to two subunits Ic (Mr = 145,000) and Sc (Mr = 130,000). Elastase and chymotrypsin are not effective. Enzymic and chemical deglycosylations of SI with endo-beta-N-acetylglucosaminidase F/glycopeptidase F and trifluoromethanesulfonic acid (TFMS) as well as probing for the binding capacity of SI to Helix pomatia lectin demonstrated that pro-SIc, Ic, and Sc are N- and O-glycosylated. Furthermore, the results were indicative of a posttranslational O-glycosylation of pro-SI, since (i) the earliest detectable precursor form, pro-SIh, did not bind to H. pomatia lectin and (ii) its deglycosylation products with both endo-beta-N-acetylglucosamidase H and TFMS were identical. Both the Sc and Ic subunits contain eight N-linked glycan units, at least one of which is of the high mannose type and found on Sc. Finally, Sc, but not Ic, was shown to display at least four populations varying in their content of O-linked glycans. The heterogeneous O-glycosylation pattern of Sc could be correlated with the distal position of this subunit (and its O-glycosylation sites) within the pro-SI molecule, thus affecting the extent of O-linked oligosaccharide processing and their subsequent presentation on the mature molecule.
...
PMID:Biosynthesis of the human sucrase-isomaltase complex. Differential O-glycosylation of the sucrase subunit correlates with its position within the enzyme complex. 336 77

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].
...
PMID:Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes. 336 55

Recently, we have characterized a membrane-bound (mb) component of brain protein carboxylmethyltransferase II (PCMT) which effectively carboxylmethylates endogenous mb methyl-accepting proteins (MAPs). (Neurochem. Int., 10 (1987) 155). We have also shown that exposing mb-MAPs to mild alkali leads to a marked increase in their recognition by PCMT. Since one of the likely consequences of the alkaline treatment appears to be the deamidation of selected protein-bound asparagines or aspartates, followed by the formation, in their place, of D-or L-isoaspartates, it is reasonable to assume that mb-MAPs constitute unique targets for the mb-PCMT because they contain such unnatural aspartate residues. Testing the relevance of this notion to the aging of cerebral mb-MAPs we focus in this report on age-related changes involving mb-MAPs. When two-or six-times washed (in 50 mM NaPO4 buffer, pH 6.5) 17,500 g, 30-min membranes or Percoll-gradient purified synaptic membranes were prepared from young (3-4 months) and old (11-12 months) rat brains and were incubated with 20 microM [3H]methyl S-adenosyl-L-methionine at pH 6.0, mb-MAP carboxyl[3H]methylation was significantly more intense in the old than in the young membranes, no additional increase being noted at 28-35 months. Mb-MAP carboxylmethylation increases were confirmed over a wide range of membrane protein concentrations and incubation times and are taken to reflect age-related modifications of the primary structure of susceptible mb-MAPs. To investigate these, we incubated young and old membranes, as well as their Lubrol-Px (1%) extracts (30 min, 0 degree C), with 0.05 M NH4OH for 90 min at 37 degrees C, a treatment which left PCMT activity largely unaffected. Our findings reveal that the effect of the NH4OH treatment on the generation of carboxylmethylatable sites was markedly smaller in "old" than in "young" proteins, suggesting that "new" carboxylmethylatable sites are generated in susceptible mb-MAPs in situ, by a process accompanying, or otherwise marking, the natural aging of neural membrane proteins.
...
PMID:The carboxylmethylation of cerebral membrane-bound proteins increases with age. 339 4

Dictyostelium discoideum synthesizes a 23,000 Mr protein, p23dd-ras, closely related to the mammalian oncogene-encoded protein p21ras. To investigate the subcellular localization of p23dd-ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23dd-ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation. In the absence of suitable protease inhibitors considerable amounts of a truncated form of p23dd-ras were recovered in the cytosol fraction, suggesting that intact p23dd-ras is attached to the membrane by a short terminal peptide sequence. Radio-isotope labelling of D. discoideum with myristic acid or palmitic acid in the presence of excess unlabelled acetate resulted in radio-isotope incorporation into a select group of proteins including p23dd-ras. No acyl label appeared in the truncated cytoplasmic form of p23dd-ras when cell breakage was performed in the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23dd-ras, like its mammalian counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30-minute pulse of methionine for a cytoplasmic precursor to the membrane-bound p23dd-ras, suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro-p21ras in mammalian cells.
...
PMID:A ras-encoded protein in Dictyostelium discoideum is acylated and membrane-associated. 344 63

Friend erythroleukemia cells, grown in the presence of dimethyl sulfoxide for 3 d, synthesize unequal amounts of the two chains (alpha and beta) of spectrin with approximately 15-30% more beta than alpha spectrin. When cells were ruptured by nitrogen cavitation, nascent alpha and beta spectrin were found to be associated with a membranous cell fraction and were not detected in the soluble cytoplasmic cell fraction. Nascent membrane-bound spectrin appeared not to be protected by membranes, since it was susceptible to trypsin degradation in the absence of detergent. On fractionation of cells with 1% Triton X-100, more (1.75-fold) nascent spectrin was found in the Triton-soluble fraction than in the Triton-insoluble fraction (cytoskeleton). In the Triton-soluble fraction, there was 55% more nascent beta spectrin than alpha spectrin, while the cytoskeleton contained nearly equal amounts of alpha and beta spectrin. Cells were pulse-labeled with L-[35S]methionine for 2 min and chase incubated for varying periods of time from 15 to 90 min with nonradioactive L-methionine. Radioactive spectrin accumulated in the Triton-soluble fraction for the first 15 min of chase incubation and then dropped by 25% in the next hour. By contrast, the amount of radioactive spectrin in the Triton-insoluble fraction rose gradually for 1 h of the chase period. This indicates that, in Friend erythroleukemia cells, a pool of membrane-bound spectrin containing an excess of the beta polypeptide is used to form the cytoskeletal system which is composed of equal molar amounts of alpha and beta spectrin. The location of spectrin was determined by immunoelectron microscopy. Small amounts of spectrin were detected in cells not treated with dimethyl sulfoxide and in these cells it was located on the surface membrane and within the cytoplasm. On treatment with dimethyl sulfoxide, complex vacuolar structures containing viruses appeared in the cells. In cells treated with dimethyl sulfoxide for 3 d 30% of the spectrin was near the outer membrane and 25% was associated with vacuolar structures, whereas in cells treated for 5 and 7 d the majority of spectrin (57-61%) was located in the vacuolar areas.
...
PMID:Biosynthesis of spectrin and its assembly into the cytoskeletal system of Friend erythroleukemia cells. 345 31

Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane. EIImtl was selectively radiolabeled with either [35S]methionine or a mixture of 14C-labeled amino acids in E. coli minicells harboring a plasmid containing the mannitol operon. The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000). The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin. This fragment was identified as the N-terminal half of the protein by comparing the amount of the original [35S]methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl. The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease. Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody. The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions. These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains. 354 89

Plasmids encoding N-terminal segments of the Escherichia coli lactose permease (also referred to as lactose carrier) have been used to analyze the biosynthesis and membrane insertion of this complex integral protein of the cytoplasmic membrane. Such truncated polypeptides were found to be stably associated with the membrane and to resemble the full-length protein with respect to their solubilization characteristics. Membrane-bound and free cytoplasmic polysomes were prepared from plasmid-bearing cells and incubated in the presence of [35S]methionine to permit completion of polypeptides initiated in vivo. Under these conditions, lactose permease was found to be radiolabeled in the fraction of membrane-bound polysomes; beta-galactosidase, used as a control, was translated almost exclusively by free polysomes. From similar experiments with N-terminal segments of lactose permease, we estimate that at most a polypeptide of 120 amino acid residues emerging from the ribosome is needed to target the nascent chain to the lipid bilayer and to mediate attachment of the ribosome to the membrane during elongation. Additional data support the idea that even shorter N-terminal sequences of 50 and 71 amino acid residues contain sufficient 'information' to provide contact with the membrane.
...
PMID:The N-terminal region of Escherichia coli lactose permease mediates membrane contact of the nascent polypeptide chain. 354 97

<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>