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Kermit L. Carraway - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and sequencing of a 58 kda membrane and microfilament associated protein from Ascites Tumor Cell microvilli with sequence similarities to retroviral gag proteins
Journal of Biological Chemistry, 1994Co-Authors: Shin-hun Juang, C. A. Carothes Carraway, Jiaqui Huang, Pedro J I Salas, Nevis Fregien, Kermit L. CarrawayAbstract:Abstract The MAT-C1 subline of the 13762 rat mammary adenocarcinoma has highly stable, branched microvilli and immobile Cell surface receptors. A membrane- and microfilament-associated 58-kDa protein (p58) in the MAT-C1 microvilli has been implicated in the stabilization of the microvilli and microfilament-membrane interactions. This protein is associated with a high M(r) glycoprotein complex containing the (proto)oncogene p185neu and other signal transduction components in a putative microfilament-associated signal transduction particle. Amino acid sequences were obtained from two trypsin peptides of p58. Screening a MAT-C1 cDNA library with a degenerate oligonucleotide derived from the larger peptide and polymerase chain reaction amplification of cDNA ends permitted the isolation of overlapping cDNAs encoding the 427-amino acid open reading frame of p58. In vitro transcription and translation using a full-length cDNA gave a protein of approximately 55 kDa, which reacts with anti-p58 antiserum and reconstitutes into a complex with actin and glycoproteins from the membrane-microfilament interaction site. When COS-7 Cells were transfected with the full-length cDNA, p58 was localized in a punctate distribution. In addition, the transfected Cells exhibited fewer microfilament cables than untransfected neighboring Cells. The amino acid sequence showed a surprising similarity to mammalian retroviral Gag proteins and included regions corresponding to p15, p12 and the N-terminal 80% of p30. Comparisons of p58 and the corresponding regions of the Gag proteins for Moloney murine leukemia virus indicated that about 60% of their amino acid residues were identical. These studies suggest that p58 is the product of an endogenous retroviral gene whose expression as a Cellular protein alters the properties of the Tumor Cell to provide a selective advantage for Tumor growth in the animal.
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Membrane-microfilament interactions in Ascites Tumor Cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex.
The Journal of biological chemistry, 1991Co-Authors: C. A. Carothes Carraway, Hua Fang, Shin-hun Juang, Yuechueng Liu, Maria E. Carvajal, Kermit L. CarrawayAbstract:[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with Ascites Tumor Cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.
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biosynthetic maturation of an Ascites Tumor Cell surface sialomucin evidence for o glycosylation of Cell surface glycoprotein by the addition of new oligosaccharides during recycling
Journal of Biological Chemistry, 1991Co-Authors: Steven R Hull, E D Sugarman, Julie Spielman, Kermit L. CarrawayAbstract:Previous biosynthetic studies of the Ascites 13762 rat mammary adenocarcinoma Cell surface sialomucin ASGP-1 (Ascites sialoglycoprotein-1) showed that it is synthesized initially as a poorly glycosylated immature form, which is converted to a larger premature form (t1/2 30 min) and more slowly to the mature glycoprotein (t1/2 greater than 4 h). In the present study O-glycosylation of ASGP-1 polypeptide is shown to occur in two phases: an early phase complete in less than 30 min, which corresponds to the synthesis of the premature form, and a later phase that continues for hours and corresponds to the synthesis of the mature form. Pulse-chase labeling studies indicate that 95% of the ASGP-1 has moved to the Cell surface in 2 h. Since transit to the Cell surface is faster than the slow phase of addition of new oligosaccharides, some new oligosaccharides must be added after ASGP-1 has reached the Cell surface. Initiation of new oligosaccharides on Cell surface ASGP-1 was demonstrated directly using a biotinylation procedure to identify Cell surface molecules. Glucosamine labeling of biotinylated ASGP-1 was shown to occur on galactosamine residues, which are linked to the polypeptide, establishing the addition of new oligosaccharides to the Cell surface molecules. Finally, resialylation studies indicate that ASGP-1 rapidly recycles through a sialylating compartment. From these results we propose that ASGP-1 reaches the Cell surface in an incompletely glycosylated state and that additional oligosaccharides are added to the glycoprotein in a second process involving recycling.
C. A. Carothes Carraway - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and sequencing of a 58 kda membrane and microfilament associated protein from Ascites Tumor Cell microvilli with sequence similarities to retroviral gag proteins
Journal of Biological Chemistry, 1994Co-Authors: Shin-hun Juang, C. A. Carothes Carraway, Jiaqui Huang, Pedro J I Salas, Nevis Fregien, Kermit L. CarrawayAbstract:Abstract The MAT-C1 subline of the 13762 rat mammary adenocarcinoma has highly stable, branched microvilli and immobile Cell surface receptors. A membrane- and microfilament-associated 58-kDa protein (p58) in the MAT-C1 microvilli has been implicated in the stabilization of the microvilli and microfilament-membrane interactions. This protein is associated with a high M(r) glycoprotein complex containing the (proto)oncogene p185neu and other signal transduction components in a putative microfilament-associated signal transduction particle. Amino acid sequences were obtained from two trypsin peptides of p58. Screening a MAT-C1 cDNA library with a degenerate oligonucleotide derived from the larger peptide and polymerase chain reaction amplification of cDNA ends permitted the isolation of overlapping cDNAs encoding the 427-amino acid open reading frame of p58. In vitro transcription and translation using a full-length cDNA gave a protein of approximately 55 kDa, which reacts with anti-p58 antiserum and reconstitutes into a complex with actin and glycoproteins from the membrane-microfilament interaction site. When COS-7 Cells were transfected with the full-length cDNA, p58 was localized in a punctate distribution. In addition, the transfected Cells exhibited fewer microfilament cables than untransfected neighboring Cells. The amino acid sequence showed a surprising similarity to mammalian retroviral Gag proteins and included regions corresponding to p15, p12 and the N-terminal 80% of p30. Comparisons of p58 and the corresponding regions of the Gag proteins for Moloney murine leukemia virus indicated that about 60% of their amino acid residues were identical. These studies suggest that p58 is the product of an endogenous retroviral gene whose expression as a Cellular protein alters the properties of the Tumor Cell to provide a selective advantage for Tumor growth in the animal.
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Membrane-microfilament interactions in Ascites Tumor Cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex.
The Journal of biological chemistry, 1991Co-Authors: C. A. Carothes Carraway, Hua Fang, Shin-hun Juang, Yuechueng Liu, Maria E. Carvajal, Kermit L. CarrawayAbstract:[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with Ascites Tumor Cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.
Shin-hun Juang - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and sequencing of a 58 kda membrane and microfilament associated protein from Ascites Tumor Cell microvilli with sequence similarities to retroviral gag proteins
Journal of Biological Chemistry, 1994Co-Authors: Shin-hun Juang, C. A. Carothes Carraway, Jiaqui Huang, Pedro J I Salas, Nevis Fregien, Kermit L. CarrawayAbstract:Abstract The MAT-C1 subline of the 13762 rat mammary adenocarcinoma has highly stable, branched microvilli and immobile Cell surface receptors. A membrane- and microfilament-associated 58-kDa protein (p58) in the MAT-C1 microvilli has been implicated in the stabilization of the microvilli and microfilament-membrane interactions. This protein is associated with a high M(r) glycoprotein complex containing the (proto)oncogene p185neu and other signal transduction components in a putative microfilament-associated signal transduction particle. Amino acid sequences were obtained from two trypsin peptides of p58. Screening a MAT-C1 cDNA library with a degenerate oligonucleotide derived from the larger peptide and polymerase chain reaction amplification of cDNA ends permitted the isolation of overlapping cDNAs encoding the 427-amino acid open reading frame of p58. In vitro transcription and translation using a full-length cDNA gave a protein of approximately 55 kDa, which reacts with anti-p58 antiserum and reconstitutes into a complex with actin and glycoproteins from the membrane-microfilament interaction site. When COS-7 Cells were transfected with the full-length cDNA, p58 was localized in a punctate distribution. In addition, the transfected Cells exhibited fewer microfilament cables than untransfected neighboring Cells. The amino acid sequence showed a surprising similarity to mammalian retroviral Gag proteins and included regions corresponding to p15, p12 and the N-terminal 80% of p30. Comparisons of p58 and the corresponding regions of the Gag proteins for Moloney murine leukemia virus indicated that about 60% of their amino acid residues were identical. These studies suggest that p58 is the product of an endogenous retroviral gene whose expression as a Cellular protein alters the properties of the Tumor Cell to provide a selective advantage for Tumor growth in the animal.
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Membrane-microfilament interactions in Ascites Tumor Cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex.
The Journal of biological chemistry, 1991Co-Authors: C. A. Carothes Carraway, Hua Fang, Shin-hun Juang, Yuechueng Liu, Maria E. Carvajal, Kermit L. CarrawayAbstract:[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with Ascites Tumor Cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.
Helmuth Hilz - One of the best experts on this subject based on the ideXlab platform.
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adp ribosylated histone h1 isolation from ehrlich Ascites Tumor Cell nuclei and partial characterization
FEBS Journal, 2005Co-Authors: Heinrichchristian Braeuer, Peter Adamietz, Ulrich Nellessen, Helmuth HilzAbstract:Conjugates between histone H1 and (ADP-ribose)n, formed in isolated nuclei of Ehrlich Ascites Tumor Cells, were purified free of unmodified H1 using perchloric acid extraction, ion-exchange and boronate-Cellulose chromatography. The isolated conjugates comprised 0.6% of the total protein-bound ADP-ribose residues, and about 1% of the histone H1 population. Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining for protein or by fluorography of the [3H]ADP-ribose residues. The neighboring bands appeared to differ from cach other by a single ADP-ribose residue. In most preparations a mean chain length of 2–3 was found. Removal of the ADP-ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher-resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non-ADP-ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP-ribosyl transfer. Studies on the lability of the (ADP-ribose)n protein linkage showed that about 20% of the ADP-ribose was linked by an NH2OH/NaOH-sensitive bond, 70% by an NH2OH-resistant/NaOH-sensitive bond, and the residual 10% apparently by an additional, NaOH-resistant bond. Cleavage of the (ADP-ribose)n- histone H1 conjugates by N-bromosuccinimide and gel eleetrophoretic analysis of the two fragments revealed that by far the most ADP-ribose residues were linked (presumably at multiple sites) to the C-terminal fragment. Furthermore, a large fraction of the conjugates carried ADP-ribosyl groups exclusively either at the C-terminal fragment or at the N-terminal fragment.
Nunez I De Castro - One of the best experts on this subject based on the ideXlab platform.
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l glutamine transport in native vesicles isolated from ehrlich Ascites Tumor Cell membranes
Journal of Bioenergetics and Biomembranes, 1991Co-Authors: Miguel Angel Medina, Ana R Quesada, Nunez I De CastroAbstract:Native vesicles isolated from Ehrlich Ascites Tumor Cells accumulate glutamine by means of Na(+)-dependent transport systems; thiocyanate seems to be the more effective anion. The apparent affinity constant for the process was 0.38 mM. The Arrhenius plot gave an apparent activation energy of 12.3 kJ/mol. The structural analogs of glutamine, acivicin (2.5 mM) and azaserine (2.5 mM), inhibited the net uptake by 67 and 70%, respectively. The sulfhydryl reagents mersalyl, PCMBS, NEM, and DTNB also inhibited net uptake, suggesting that sulfhydryl groups may be involved in the activity of the carrier protein. A strong inhibition was detected when the vesicles were incubated in the presence of alanine, cysteine, or serine; in addition, histidine, but not glutamate or leucine, had a negative effect on glutamine transport.
