scholarly journals Preparation of hepatic gap (communicating) junctions. Identification of the constituent polypeptide subunits

1977 ◽  
Vol 168 (3) ◽  
pp. 475-481 ◽  
Author(s):  
Janetta G. Culvenor ◽  
W. Howard Evans
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Density Perturbation ◽  
Morphological Properties ◽  
Communicating Junctions ◽  
Transmembrane Channels ◽  
A Minor

1. Gap (communicating) junctions are plasma-membrane specializations of characteristic morphology that form transmembrane channels allowing direct communication between cells. Their preparation is described starting from mouse liver plasma membranes and the constituent polypeptides are deduced. 2. Gap junctions co-purify with collagen fibres when the plasma-membrane residues insoluble in N-dodecyl sarcosinate are fractionated on sucrose gradients. Sucrose-density perturbation by relipidation of isolated gap junctions or the use of urea to remove non-junctional membranes both failed to diminish the collagen content of fractions. 3. Removal of collagen by treatment with purified collagenase preparations yielded morphologically satisfactory gap-junction fractions. Analysis by polyacrylamide-gel electrophoresis of the polypeptides present in gap junctions prepared by procedures omitting or using collagenases indicated two non-glycosylated polypeptides, a major component of apparent mol.wt. 38000 and a minor 40000-mol.wt. component. These two polypeptides were also present in plasma membranes and the intermediate fractions. 4. Proteolysis of the gap-junction polypeptides yielding components of mol.wt. 34000, 25000 and below 20000 occurred when iodinated gap junctions were subject to prolonged collagenase treatment, thus explaining the variable polypeptide composition of gap junctions reported by others. 5. The morphological properties of the isolated gap junctions prepared by the various procedures are described.

Dissociation of lens fibre gap junctions releases MP70

1988 ◽  
Vol 91 (3) ◽  
pp. 415-421 ◽  
Author(s):  
J. Kistler ◽  
S. Bullivant
Keyword(s):  
Electron Microscope ◽  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Membrane Structures ◽  
Treatment Results ◽  
Double Membrane ◽  
Lens Fibre ◽  
Junction Proteins

MIP and MP70 are putative gap junction components in the plasma membranes of the mammalian lens fibre cells. We show now that MP70 can be solubilized separately from MIP in mild detergent solutions, and that this treatment results in the dissociation of the fibre gap junctions. Solubilized MP70 was isolated as 16.9 S particles by velocity gradient centrifugation and in the electron microscope had the appearance of short double-membrane structures consistent with connexon-pairs. These observations open a new experimental avenue in which to characterize separately the two putative lens gap junction proteins structurally and functionally.

Heart gap junction preparations reveal hemiplaques by atomic force microscopy

1995 ◽  
Vol 268 (4) ◽  
pp. C968-C977 ◽  
Author(s):  
R. Lal ◽  
S. A. John ◽  
D. W. Laird ◽  
M. F. Arnsdorf
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Long Range ◽  
Connexin 43 ◽  
Plasma Membranes ◽  
Aqueous Media ◽  
Freeze Fracture ◽  
Isolated Rat Heart ◽  
Hexagonal Packing ◽  
Atomic Force

Current structural models of gap junctions indicate two apposed plasma membranes with hexagonally packed hemichannels in each membrane aligning end to end. These channels connect the cytoplasms of contacting cells. Images of isolated rat heart gap junctions have been made with the atomic force microscope in aqueous media. We show that native cardiac gap junctions have a thickness of 25 +/- 0.6 nm. This decreases to 17 nm when they are treated with trypsin, which is known to remove some cytoplasmic components of connexin 43. Imaging shows subunits with a center to center spacing of approximately 9-10 nm and long range hexagonal packing, measurements in agreement with studies using freeze-fracture and negative-stain electron microscopy. In addition to gap junctions, we imaged structures that had all the characteristics of native gap junctions except their thickness was limited to 9-11 nm. They also show long range hexagonal packing and center to center spacing of 9-10 nm. These structures decrease in thickness, to 6-9 nm, when treated with trypsin. We have called these structures hemiplaques. They appear to be present endogenously in the preparation, as we have ruled out their being an artifact of imaging by AFM. However, it remains to be determined if they are a consequence of the procedure used in isolating gap junctions or a possible intermediary in gap junction formation.

scholarly journals Paralemmin, a Prenyl-Palmitoyl–anchored Phosphoprotein Abundant in Neurons and Implicated in Plasma Membrane Dynamics and Cell Process Formation

1998 ◽  
Vol 143 (3) ◽  
pp. 795-813 ◽  
Author(s):  
Christian Kutzleb ◽  
Gabriele Sanders ◽  
Raina Yamamoto ◽  
Xiaolu Wang ◽  
Beate Lichte ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Species Conservation ◽  
Cell Types ◽  
Membrane Dynamics ◽  
Morphological Properties ◽  
Developmentally Regulated ◽  
Membrane Flow ◽  
Western Blots ◽  
Process Formation

We report the identification and initial characterization of paralemmin, a putative new morphoregulatory protein associated with the plasma membrane. Paralemmin is highly expressed in the brain but also less abundantly in many other tissues and cell types. cDNAs from chicken, human, and mouse predict acidic proteins of 42 kD that display a pattern of sequence cassettes with high inter-species conservation separated by poorly conserved linker sequences. Prenylation and palmitoylation of a COOH-terminal cluster of three cysteine residues confers hydrophobicity and membrane association to paralemmin. Paralemmin is also phosphorylated, and its mRNA is differentially spliced in a tissue-specific and developmentally regulated manner. Differential splicing, lipidation, and phosphorylation contribute to electrophoretic heterogeneity that results in an array of multiple bands on Western blots, most notably in brain. Paralemmin is associated with the cytoplasmic face of the plasma membranes of postsynaptic specializations, axonal and dendritic processes and perikarya, and also appears to be associated with an intracellular vesicle pool. It does not line the neuronal plasmalemma continuously but in clusters and patches. Its molecular and morphological properties are reminiscent of GAP-43, CAP-23, and MARCKS, proteins implicated in plasma membrane dynamics. Overexpression in several cell lines shows that paralemmin concentrates at sites of plasma membrane activity such as filopodia and microspikes, and induces cell expansion and process formation. The lipidation motif is essential for this morphogenic activity. We propose a function for paralemmin in the control of cell shape, e.g., through an involvement in membrane flow or in membrane–cytoskeleton interaction.

scholarly journals Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions

2002 ◽  
Vol 365 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Translation System ◽  
Alternative Mechanism ◽  
Protein Cleavage ◽  
Membrane Components ◽  
Gap Junction Hemichannels

Gap-junction channels provide a widespread intercellular signalling mechanism. They are constructed of a family of connexin membrane proteins that thread across the membrane four times and oligomerize to generate hexameric gap-junction hemichannels. Using an in vitro cell-free transcription/translation system, we demonstrate that connexin (Cx) 26, one of the smallest connexins, is integrated directly in a post-translational manner into plasma membranes. Protein-cleavage studies of Cx26 integrated into plasma membranes indicate a similar native transmembrane topography to that of Cx26 integrated co-translationally into microsomes. Cx26 integrated post-translationally into plasma membranes oligomerizes and, when incorporated into liposomes, provides permeability to ascorbic acid, suggesting that gap-junction hemichannels are generated. The results provide the basis of a novel alternative mechanism for spontaneous assembly in plasma membranes of Cx26 gap-junction hemichannels that occurs independently of the conventional biogenesis of gap junctions involving connexin trafficking and oligomerization via membrane components of the secretory pathway.

scholarly journals On gap junction structure.

1980 ◽  
Vol 86 (1) ◽  
pp. 190-198 ◽  
Author(s):  
G Zampighi ◽  
J M Corless ◽  
J D Robertson
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rat Liver ◽  
Extracellular Space ◽  
Plasma Membranes ◽  
Negative Staining ◽  
The Other ◽  
Central Axis ◽  
Thin Sectioning ◽  
Junction Structure

We have studied the stain distribution within rat liver gap junctions for specimens prepared by thin sectioning and negative staining. Pools of stain molecules exist in two specific locations with respect to the distinctive morphological units (connexons) of the junction. One pool of stain surrounds the connexons and is restricted to the extracellular space in the gap between the adjacent plasma membranes. The other pool of stain is located along in the central axis of each connexon, measures 1-2 nm in diameter and 4-5 nm in length, and is restricted to the gap region. On rare occasions, barely discernible linear densities seem to extend from this latter pool of stain and traverse the entire width of the junction. The data indicate the existence of a hydrophilic cavity along the central axis of te connexon which, in most instances, is restricted to the gap region. However, the precise depth to which this cavity may further extend along the connexon axis is still uncertain.

scholarly journals Connexin 43 K63-polyubiquitination on lysines 264 and 303 regulates gap junction internalization

2017 ◽  
Author(s):  
Rachael M. Kells-Andrews ◽  
Rachel A. Margraf ◽  
Charles G. Fisher ◽  
Matthias M. Falk
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Room Temperature ◽  
Cell Communication ◽  
Mapk Phosphorylation ◽  
Annular Gap ◽  
Summary Statement ◽  
Triton X

ABSTRACTGap junctions (GJs) assembled from connexin (Cx) proteins play a pivotal role in cell-to-cell communication by forming channels that connect the cytosols of adjacent cells. Connexin 43, the best-studied Cx, is ubiquitously expressed in vertebrates. While phosphorylation is known to regulate multiple aspects of GJ function, much less is known about the role ubiquitination plays in these processes. Here we show by using ubiquitination-type specific antibodies and Cx43 lysine (K) to arginine (R) mutants that a portion of Cx43 in GJs can become K63-polyubiquitinated on K264 and K303. Relevant Cx43 K/R mutants assembled significantly larger GJ plaques, exhibited much longer protein half-lives and were internalization impaired. Interestingly, ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitination may be triggered by phosphorylation. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK phosphorylation sites. Together, these novel findings suggest that upon internalization, some Cx43 in GJs becomes K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination may be induced by Cx phosphorylation.Summary StatementHere we show that connexin 43 in gap junctions becomes K63-poly ubiquitinated on lysines 264 and 303 and its requirement for gap junction endocytosis. These novel findings significantly contribute to our understanding of GJ turnover and patho-/physiology.Abbreviations usedAGJannular gap junctionAMSHassociated molecule with the SH3 domain of STAMCMEclathrin-mediated endocytosisCxConnexinCx43Connexin 43DUBdeubiquitinaseGJgap junctionMonoUbmonoubiquitinNedd4-1neural precursor cell expressed developmentally down-regulated protein 4-1PMplasma membranePolyUbpolyubiquitinTPA12-O-Tetradecanoylphorbol 13-AcetateTX-100Triton X-100RTroom temperatureUbubiquitin

Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

1990 ◽  
Vol 258 (1) ◽  
pp. C179-C184 ◽  
Author(s):  
G. Schmalzing ◽  
P. Eckard ◽  
S. Kroner ◽  
H. Passow
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Sodium Pumps

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by [gamma-32P]ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from [3H]ouabain bound to the cell surface before maturation could be phosphorylated with inorganic [32P]phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

scholarly journals Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase-catalysed iodination and isolation after density perturbation with digitonin

1975 ◽  
Vol 152 (1) ◽  
pp. 71-84 ◽  
Author(s):  
B A Lewis ◽  
A Elkin ◽  
R H Michell ◽  
R Coleman
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Density Perturbation ◽  
Subcellular Fractionation ◽  
Mitochondrial Fraction ◽  
Intestinal Epithelial ◽  
Isolated Cells ◽  
Cell Sheets ◽  
Basolateral Plasma Membrane ◽  
Membrane Bound

1. Lactoperoxidase-catalysed iodination was used to label intestinal epithelial cell sheets with 125I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane-bound 125I therefore provided an effective marker for following plasma-membrane fragments through subcellular-fractionation procedures. 2. After homogenization and isopycnic zonal centrifugation through sucrose gradients two peaks of membrane-bound 125I were detected. One coincided with brush border enzymes such as alkaline phosphatase, disaccharidases and L-leucine β-naphthylamidase, whereas the other was coincident with the major peak of (Na++K+)-stimulated ATPase (adenosine triphosphatase), which has been thought to be concentrated in the basolateral plasma membranes of these cells. Neither peak of 125I reflected the distribution of any marker for an intracellular organelle. 3. A larger proportion of the (Na++K+)-stimulated ATPase, and thus of the basolateral plasma-membrane material, was found in a crude ‘mitochondrial’ fraction. It was not readiily separated from mitochondria by conventional techniques of subcellular fractionation. 4. Treatment of the ‘mitochondrial’ fraction with digitonin increased the density of basolateral plasma membrane but had little effect on mitochondrial density. A purified preparation of digitonin-loaded basolateral plasma membranes was isolated at a density of 1.20-1.22 by isopycnic centrifugation. 5. The enzymic composition of this preparation of basolateral plasma membranes is compared with previous preparations isolated from intestinal mucosal ‘scrape’ materials and from isolated cells.

A biochemical dissection of the cardiac intercalated disk: isolation of subcellular fractions containing fascia adherentes and gap junctions

1981 ◽  
Vol 52 (1) ◽  
pp. 313-325
Author(s):  
C.A. Colaco ◽  
W.H. Evans
Keyword(s):  
Gap Junctions ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intercellular Junctions ◽  
Blue Staining ◽  
Molecular Weights ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Insoluble Material ◽  
Rats And Mice

In view of our limited knowledge of the biochemical composition of intercellular junctions, a method was developed for the preparation from rats and mice of plasma membranes containing cardiac intercalated disks. When these membranes were extracted with detergents, e.g. N-lauryl sarcosinate or deoxycholate, the detergent-insoluble material contained structures derived mainly from fascia adherentes junctions, but a few gap junctions and maculae adherentes were also present. When the detergent extraction was carried out at an alkaline pH, the maculae adherentes junctions were dissolved. Fractionation of the detergent-insoluble extract on a sucrose gradient yielded a fraction containing fascia adherentes junction of density 1.20-1.26 g/cm3. Gap junctions banded at a lower density, 1.16-1.20 g/cm3. Polyacrylamide gel electrophoresis showed that the major polypeptide bands in the fascia adherentes-enriched fraction were of molecular weights 134000, 108000, 62–64000, 58000, 47000 and 43000. Although fractions with the gap junctions were contaminated by fascia adherentes junctions, the major polypeptides were calculated by subtraction to be of mol. wt 37000, 26000 and 19000. Two glycoproteins corresponding to minor polypeptides visualized by Coomassie Blue staining were present in the fascia adherentes fraction. Comparison of the fascia adherentes-enriched fraction with a Z-disc fraction prepared from rabbit hearts indicated a different morphology and polypeptide composition.

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