Independent lines of evidence suggesting a major gap junctional protein with a molecular weight of 26,000.

Finbow, Malcolm E.; Yancey, S B; Johnson, Randy; Revel, Jean‐Paul

doi:10.1073/pnas.77.2.970

Cited by 62 publications

(36 citation statements)

References 14 publications

(13 reference statements)

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“…wt. 26 000) of lens bound to isolated lens junctions, Paul and Goodenough (1983) showed that similar antisera bound only to non- consistent from a given source (e.g., 1-2 /4g/g wet weight mouse liver no matter whether starting from a crude homogenate or a post-mitochondrial pellet) and varies in preparations from different sources, as would be expected from freeze fracture studies; liver > V79 cells > BHK cells (Yancey et al, 1978(Yancey et al, , 1982Revel et al, 1971 (Hertzberg and Gilula, 1979;Henderson et al, 1979;Finbow et al, 1980;Nicholson et al, 1981;Traub et al, 1982) or this latter may be a dimeric form of the 16 K protein. This does not appear to be the answer, at least in the case of the 28 K protein isolated by Nicholson et al (1981).…”

Section: Discussionmentioning

confidence: 93%

“…Under these conditions, nuclei and cytoskeletal elements remain intact and can thus be removed by low speed centrifugation. The procedure then uses adaptations of existing techniques to remove contaminating proteins (Finbow et al, 1980) (Finbow, Lane, Shuttleworth and Pitts, submitted). The method has been applied to mouse (Figures 3 and 4), rat ( Figure 11) and chicken liver, mouse heart, brain and uterus and to cultured BRL cells (Figures 3 and 4 during purification is electron microscopy.…”

Section: Resultsmentioning

confidence: 99%

“…wt. of 26 000-28 000 (Hertzberg and Gilula, 1979;Henderson et al, 1979;Finbow et al, 1980;Nicholson et al, 1981). Although a number of laboratories have reported the 26-28 K protein to be the major constituent of liver gap junctions, recent studies in Goodenough's laboratory failed to detect this as a major component in morphologically pure junction fractions prepared from mouse liver or mouse heart in the absence of added proteases (Kensler and Goodenough, 1980; Fallon and Goodenough, 1981).…”

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Analysis of vertebrate gap junction protein.

et al. 1983

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confidence: 93%

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confidence: 99%

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Analysis of vertebrate gap junction protein.

et al. 1983

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“…Gap junctions can be isolated by subcellular fractionation following alkali or detergent treatment (10,11). While there are some discrepancies, it is generally agreed that the constituent protein is z27 kDa (cf.…”

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cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide.

Sáez

Spray²,

Nairn

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

341

145

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Membrane-permeant cAMP derivatives (dibutyryl-and 8-bromo-cAMP) increase gap-junctional conductance within minutes when applied to voltage-clamped pairs of rat hepatocytes. Glucagon also increases junctional conductances, but the response has a more rapid onset and is more rapidly reversible. The glucagon effect can be prevented by intracellular injection of the protein inhibitor of the cAMPdependent protein kinase (Walsh inhibitor), indicating that the catalytic subunit of cAMP-dependent protein kinase is directly involved. The 27-kDa miijor gap junction polypeptide is phosphorylated when liver cells dissociated into small groups are incubated with 32p. Addition of 8-bromo-cAMP to cells increases the incorporation of 32P into the 27-kDa junctional protein. Serine is the amino acid residue that is phosphorylated. When isolated liver gap junctions are incubated in the presence of catalytic subunit of the cAMP-dependent protein kinase, the 27-kDa gap junction polypeptide is phosphorylated with low stoichiometry on serine. The rapid increases in gap junctional conductance caused by agents that elevate cAMP and phosphorylation of the gap junction protein by cAMPdependent protein kinase suggest that cAMP-dependent phosphorylation of the gap junction channel modulates the conductance of liver gap junctions.

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“…No such studies have been reported yet, although much of the present controversy regarding the protein components of functional gap junctions [30] could be resolved if functional reconstitution could be demonstrated.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Willecke¹,

Müller²,

Drüge³

et al. 1983

Experimental Cell Research

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SUMMARYChinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., met-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-CAMP). The isolated Chinese hamster CI-4 cells which expressed the met-phenotype most stringently showed the following characteristics:1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the met+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (met'). Confluent CI-4 cells grown in the presence of 1 mM db-CAMP showed 9% coupled cells.2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The met+ parental cells showed small gap junction plaques (0.013 % of the total cell surface analyzed).3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92 % positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic libroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68 %. Other cells of spontaneous met-phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix.4. The met-defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of met-, as well as met+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the met-defect.Most cells in organs and in tissue culture show cell-cell coupling via gap junctions which-can be demonstrated by the spreading of electrical signals, injected fluorescent dyes or radioactively labelled metabolites between contiguous cells [l, 21. In many cases gap junction plaques, i.e., arrays of particles of polygonal symmetry, have been demonstrated by different techniques of electron microscopy on apposed membranes of contiguous cells. Since the appearance of gap junction plaques and the demonstration of cell-cell coupling (by one or more of

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Independent lines of evidence suggesting a major gap junctional protein with a molecular weight of 26,000.

Cited by 62 publications

References 14 publications

Analysis of vertebrate gap junction protein.

Analysis of vertebrate gap junction protein.

cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide.

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

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