scholarly journals Gangliosides and sialosylglycoproteins in coated vesicles from bovine brain

1985 ◽  
Vol 225 (3) ◽  
pp. 713-721 ◽  
Author(s):  
D Gravotta ◽  
H J F Maccioni
Keyword(s):  
Rat Brain ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Molar Ratio ◽  
Coated Vesicle ◽  
Synaptic Plasma Membranes ◽  
Morphological Criteria

The content of gangliosides and sialosylglycoproteins was investigated in a coated-vesicle-enriched fraction prepared from bovine brain by the method of Pearse [(1975) J. Mol. Biol. 97, 93-98] and further purified by g.p.c. (glass-permeation chromatography) [Pfeffer & Kelly (1981) J. Cell Biol. 91, 385-391]. From morphological criteria and from the analysis of the polypeptide pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the coated-vesicle fraction (CV-fraction) appeared more than 95% pure. The ganglioside-NeuAc (N-acetylneuraminate), glycoprotein-NeuAc, phospholipid and cholesterol contents of CV-fraction were compared with those of bovine brain synaptic plasma membranes (SPM). The cholesterol to phospholipid molar ratio was 0.47 +/- 0.07 in CV-fraction and 1.06 +/- 0.08 in SPM. The ganglioside-NeuAc and glycoprotein-NeuAc to phospholipid molar ratios were 0.047 and 0.020 respectively in CV-fraction and 0.039 and 0.016 respectively in SPM. The (Na+ + K+)-dependent ATPase activity sensitive to ouabain (in mumol of Pi/h per nmol of phospholipid) was 1.04 in CV-fraction and 0.63 in SPM; the ratio between this activity and the activity resistant to ouabain was 2 in CV-fraction and 1.4 in SPM. A t.l.c. analysis of the ganglioside fractions showed that most of the ganglioside species present in SPM were present in CV-fraction. In a rat brain coated-vesicle preparation not subjected to g.p.c., the activities [as sugar-radioactivity (c.p.m.) transferred/h per mumol of phospholipid] of the enzymes CMP-NeuAc:sialosyl-lactosylceramide (GM3) sialosyl-, UDP-Gal:N-acetylgalactosaminyl(sialosyl)lactosylceramide (GM2) galactosyl- and UDP-GalNAc:sialosyl-lactosylceramide (GM3) N-acetylgalactosaminyl-transferases, which were considered Golgi-apparatus markers, were about 19, 16 and 10% respectively of those determined in rat brain neuronal perikaryon-enriched fractions. Taken together, the results indicate that most of the major gangliosides are constituents of coated vesicles.

scholarly journals Rabbit reticulocyte coated vesicles carrying the transferrin- transferrin receptor complex: I. Purification and partial characterization

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1035-1039
Author(s):  
HR Choe ◽  
ST Moseley ◽  
J Glass ◽  
MT Nunez
Keyword(s):  
Transferrin Receptor ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Receptor Complex ◽  
Assembly Factor ◽  
Excellent Source

Coated vesicles bearing the transferrin-transferrin receptor complex were isolated from rabbit reticulocytes by freeze-thaw cell lysis, followed by differential centrifugation with pelleting of vesicles at 100,000 g. Electronmicroscopy demonstrated the vesicles to have the characteristic morphology of coated vesicles, including the appearance of triskelions. The protein composition of the vesicles as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis included transferrin, transferrin receptor, and proteins of apparent mol wt of approximately 180,000, 140,000, 100,000, and 47,000 daltons. The 180,000 and 100,000 mol wt proteins were identified as clathrin and coated vesicle assembly factor proteins, respectively, by Western blot analyses. The vesicles had a Mg2+-dependent ATPase with a specific activity of approximately 8.5 nmoles ATP converted/min/mg vesicle protein. The vesicles could acidify the intravesicular space, as evidenced by the stimulation of the Mg2+-ATPase by the protonophore FCCP. Reticulocytes appear to be an excellent source of coated vesicles and as such should provide a model for studying the endocytosis of transferrin and the steps of iron uptake that proceed in these vesicles.

Differeniated regions of human placental cell surface associated with exchange of materials between maternal and foetal blood: coated vesicles

1977 ◽  
Vol 25 (1) ◽  
pp. 293-312 ◽  
Author(s):  
C.D. Ockleford ◽  
A. Whyte
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Placental Tissue ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Major Protein ◽  
Membrane Components

Coated vesicles may be an important component of the micropinocytic system of the human placenta. Regions of very dense reaction with glycocalyx stains are restricted to membranes within forming and fully formed coated vesicles. This is interpreted as evidence against permanently grouped specific binding sites having a role in the selective uptake of materials by micropinocytosis, and as support for theories of coated-vesicle formation which take into account the dynamic nature of membrane components. The pyroantimonate precipitation technique which was employed in an attempt to localize cations in placental tissue at term resulted in the deposition of electron-dense material in coated vesicles and basement membrane. Examination of the distribution of coated vesicles in placental tissue explants at 8–12 weeks of gestation revealed a restricted distribution of these organelles. Probably more than 89% of coated vesicles lie within the largest vesicles' diameter from the cell surface. Placental coated vesicles were isolated and examined using negative staining. A polygonally patterened structure was apparent on their surfaces. Analysis of the isolated fraction of coated vesicles using sodium dodecyl sulphate polyacrylamide gel electrophoresis shows the presence of a major protein of molecular weight 180000. This is the same molecular weight that has been given for clathrin, the major protein of the raised polygonally patterned structure on the cytoplasmic surface of coated vesicles from other sources.

scholarly journals Rabbit reticulocyte coated vesicles carrying the transferrin- transferrin receptor complex: I. Purification and partial characterization

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1035-1039 ◽  
Author(s):  
HR Choe ◽  
ST Moseley ◽  
J Glass ◽  
MT Nunez
Keyword(s):  
Transferrin Receptor ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Receptor Complex ◽  
Assembly Factor ◽  
Excellent Source

Abstract Coated vesicles bearing the transferrin-transferrin receptor complex were isolated from rabbit reticulocytes by freeze-thaw cell lysis, followed by differential centrifugation with pelleting of vesicles at 100,000 g. Electronmicroscopy demonstrated the vesicles to have the characteristic morphology of coated vesicles, including the appearance of triskelions. The protein composition of the vesicles as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis included transferrin, transferrin receptor, and proteins of apparent mol wt of approximately 180,000, 140,000, 100,000, and 47,000 daltons. The 180,000 and 100,000 mol wt proteins were identified as clathrin and coated vesicle assembly factor proteins, respectively, by Western blot analyses. The vesicles had a Mg2+-dependent ATPase with a specific activity of approximately 8.5 nmoles ATP converted/min/mg vesicle protein. The vesicles could acidify the intravesicular space, as evidenced by the stimulation of the Mg2+-ATPase by the protonophore FCCP. Reticulocytes appear to be an excellent source of coated vesicles and as such should provide a model for studying the endocytosis of transferrin and the steps of iron uptake that proceed in these vesicles.

scholarly journals Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin

1993 ◽  
Vol 13 (4) ◽  
pp. 2061-2068
Author(s):  
H Shirataki ◽  
K Kaibuchi ◽  
T Sakoda ◽  
S Kishida ◽  
T Yamaguchi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Target Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Reading Frame ◽  
Gtp Binding ◽  
Putative Target

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis

1982 ◽  
Vol 152 (2) ◽  
pp. 616-625
Author(s):  
D J Mancuso ◽  
T H Chiu
Keyword(s):  
Thin Layer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Lipoteichoic Acid ◽  
Molar Ratio ◽  
Streptococcus Sanguis ◽  
Chromatographic Profile ◽  
Hydrolysis Of ◽  
Cellulose Column

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.

185 EXPRESSION AND LOCALIZATION OF µ-OPIOID RECEPTOR IN CANINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 172 ◽  
Author(s):  
L. Pavone ◽  
M. Albrizio ◽  
R. Minoia
Keyword(s):  
Opioid Receptor ◽  
Room Temperature ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Negative Control ◽  
Primary Antibody ◽  
Primary Role ◽  
Secondary Antibody ◽  
Rabbit Igg

Endogenous opioid peptides (EOP), through G-protein-coupled receptors, control metabolism and many physiological and pathological conditions. Once EOP are linked to their receptors, above all µ-opioid receptor (MOR), a block of the Ca2+ channel occurs (Sciorsci et al. 2000 Immunopharm. Immunotox. 22, 575–626). The disruption of Ca2+ homeostasis interferes with many Ca2+-mediated/dependent actions. Our previous studies demonstrated the presence of MOR in human, bovine, and equine oocytes, in sperm cells of several species (equine, canine, etc.), in mare's tube, in ovine, bovine and mouse embryos. The presence of MOR on the male canine gamete lets us hypothesize its presence on the female gamete, too. In this study we demonstrated the presence of MOR on canine oocytes by immunofluorescence (IF) and western blot (WB) analysis, and we speculate on its possible functional role. Canine ovaries were obtained from healthy bitches randomly chosen among those arriving at our veterinary hospital for surgical ovariectomy without considering the period of their reproductive cycle. Oocytes were collected by ovary slicing and tested to check for the presence of MOR. For IF, oocytes were washed in 100 mm glycine in PBS and incubated for 30 min in PBS-1% BSA. Control oocytes were incubated with primary rabbit polyclonal antibody against the rat 3rd extracellular loop of MOR (Chemicon, Temecula, CA, USA). All oocytes were incubated for 2 h at room temperature with a FITC-conjugated anti-rabbit IgG-secondary antibody diluted 1:200 in Evans blue/PBS, washed, and visualized by laser scanning confocal microscope. For the WB, crude plasma membranes were obtained from pools of oocytes. They were lysed in Laemli buffer and loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. After electrophoresis, proteins were electrotransferred (semi-dry apparatus, BioRad, Milano, IT) to Immobilon-P membranes (Millipore, Bedford, MA, USA). Filters were blocked for 1 h and blotted overnight at 4�C against the same primary antibody used for IF, diluted 1:7500 in blocking buffer. After washing, membranes were incubated with a 1:10 000 dilution of peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 2 h at room temperature. Reactive bands were visualized by Supersignal West Pico Chemiluminescent substrate (Pierce, Milano, IT). A negative control was performed. The IF highlighted, by clear brilliant green, the MOR's localization on canine oocytes. The negative control did not present any fluorescent region or spotted coloring. The WB revealed the presence of one immunoreactive band of approximately 65 kDa, thus confirming the results obtained by IF. No reactivity was evident when the primary antibody was adsorbed with an excess of immunizing peptide. The presence of MOR on canine oocytes indicates its possible role in the modulation of oocyte metabolism. These data strongly confirm previous evidence from our research unit on the involvement of the opioidergic system during gamete development and interaction, thus allowing us to speculate on a primary role of MOR in controlling key events of the reproductive activity.

Cross-linking of neuropeptide Y to its receptor on rat brain membranes

1989 ◽  
Vol 256 (3) ◽  
pp. G637-G643 ◽  
Author(s):  
P. J. Mannon ◽  
I. L. Taylor ◽  
L. M. Kaiser ◽  
T. D. Nguyen
Keyword(s):  
Neuropeptide Y ◽  
Rat Brain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Brain Membranes ◽  
Npy Receptor ◽  
Rat Brain Membranes ◽  
Immature Form ◽  
Triton X

The receptor for neuropeptide Y (NPY) was identified on rat brain membranes after covalent labeling with 125I-NPY using the homobifunctional cross-linkers disuccinimido suberate and disuccinimido dithiobis(propionate) and the heterobifunctional photoactive cross-linker succinimido 4-azidobenzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of two bands at Mr 62,000 and 39,000. Both species showed the same high affinity for 125I-NPY. Exposure to reducing agents did not change the migration of these bands. When the NPY receptor complex was solubilized from the membranes with 1% Triton X-100 and analyzed by gel filtration chromatography, it eluted from a Fractogel TSK 55F column as a peak at approximately 65 kDa. This peak was asymmetric with a shoulder of radioactivity that probably reflects the smaller receptor species. These data indicate that the NPY receptor on rat brain membranes is a monomeric 58-kDa unit (62 kDa minus the mass of the cross-linked NPY) without covalently or noncovalently linked subunits. The smaller 39-kDa species may be an immature form of the 62-kDa species, a second distinct receptor, or a degradation product of the 62-kDa band.

Sign in / Sign up

Export Citation Format

Share Document