Malcolm E. Finbow

Isolation and characterisation of arthropod gap junctions

Gap junctions have been isolated from the hepatopancreas of the crustacean arthropod, Nephrops norvegicus (Norway lobster). SDS‐PAGE of these preparations shows two major protein bands, mol. wt. 18 000 (18 K) and mol. wt. 28 000 (28 K). The 18‐K and 28‐K proteins are interconvertible, cannot be distinguished by two dimensional tryptic and chymotryptic peptide mapping, and therefore appear to be different (most likely monomeric and dimeric) forms of the same protein. The protein can also aggregate to higher multimeric forms mol. wt. 38 000 (presumed trimer), and mol. wt. 52 000 (presumed tetramer). The buoyant density of the isolated gap junctions in continuous potassium iodide gradients is 1.260 g/cm3. The junctions are progressively solubilized in increasing SDS concentrations, mostly between 0.1% and 0.2% SDS, and this is accompanied by the release of the 18‐K and 28‐K forms of the junctional protein. The Nephrops hepatopancreas 18‐K junctional protein has antigenic determinants in common with the vertebrate 16‐K junctional protein as shown by cross‐reactivity with two different affinity purified antibody preparations. However, no detectable similarity can be seen between the major 125I‐labelled tryptic and chymotrytpic peptides of the Nephrops hepatopancreas 18‐K protein and the mouse liver 16‐K protein.

Metabolic interactions between animal cells through permeable intercellular junctions

Abstract In mixed cultures of HGPRT− (lacking hypoxanthine: guanine phosphoribosyltransferase activity) and wild-type mammalian cells coupled by permeable intercellular junctions, the purine nucleotide pools equilibrate between the two cell types prior to incorporation into nucleic acid. As a consequence, in the presence of exogenous hypoxanthine, the mutant cells stimulate the activity of HGPRT pathway in the wild-type cells and the de novo pathway of purine nucleotide biosynthesis is inhibited in both cell types. That is, the metabolic activity of a mixed culture of coupled cells is a unique characteristic of the mixture, not simply the sum of the activities of the component cells, whereas the activity of a mixed culture of cells which are not coupled by permeable intercellular junctions is simply the sum of the activities of the two cell types.

Analysis of vertebrate gap junction protein.

A new method for the purification of gap junctions is described which depends on the extraction of cell monolayers or tissue homogenates with Triton X‐100. The major band on SDS‐polyacrylamide gel electrophoresis (PAGE) of junctional preparations from a variety of vertebrate sources has an apparent mol. wt. of 16,000 (16 K). Further evidence for the junctional origin of the 16 K protein is provided by the results of four different experimental approaches. (i) The junctions form a sharp band in potassium iodide density gradients at 1.195 g/cm3 and the 16 K protein is the only detectable band in fractions of this bouyant density. (ii) The junctions are progressively solubilised by increasing concentrations of SDS (in the range 0.1‐0.5%) and the dissolution of the junctional structure, observed by electron microscopy, parallels the release of the 16 K protein. (iii) Glutaraldehyde fixation of intact junctions cross‐links the 16 K protein. (iv) The recoverable amount of the 16 K protein correlates with known changes in gap junctional area in the regenerating weanling rat liver after partial hepatectomy and in V79 cell cultures exposed to 4beta‐phorbol 12‐myristate 13‐acetate.

The gap junction-like form of a vacuolar proton channel component appears not to be an artifact of isolation: An immunocytochemical localization study☆

Gap junctional structures containing a 16-kDa intrinsic membrane protein have been isolated from the hepatopancreas of the crustacean Nephrops norvegicus. These structures are double membranes 14-15 nm thick and composed of hexagonal arrays of particles which have a central pore that is penetrated by a cationic negative stain. Membrane preparations have also been isolated from the hepatopancreas and these contain similar gap junctional regions of uniform width. Affinity purified antibodies to the 16-kDa protein bind principally to these gap junctional regions. Antiserum raised against the isolated gap junctional structures binds strongly to the lateral surfaces of the columnar epithelial cells and in particular to gap junction-like regions.

Binding of bovine papillomavirus type 4 E8 to ductin (16K proteolipid), down-regulation of gap junction intercellular communication and full cell transformation are independent events

The E8 open reading frame of bovine papillomavirus type 4 encodes a small hydrophobic polypeptide that contributes to primary cell transformation by conferring to cells the ability to form foci and to grow in low serum and in suspension. Wild-type E8 binds in vitro to ductin, a component of gap junctions, and this binding is accompanied by a loss of gap junction intercellular communication in transformed bovine fibroblasts. However, through the analysis of a panel of E8 mutants, we show here that binding of E8 to ductin is not sufficient for down-regulation of gap junction communication and that there is no absolute correlation between down-regulation of gap junction communication and the transformed phenotype.

Junctional Permeability and Its Consequences

For the last 100 years attention has been focused on the cell as the unit of life, and indeed this important concept has formed the basis for much of our understanding of structure and function in biological systems. Although this concept is meaningful and accurate for prokaryotes and other unicellular organisms, the cell in a highly developed multicellular organism must lose much of its independence in the milieu of the composite tissues. We must now accept that multicellular organisms are populations of interacting and interdependent cells with properties which cannot be assigned specifically to individual units but instead must be collectively assigned to all the different cells which together are the developing embryo or the adult tissue.

Permeability of junctions between animal cells. Intercellular exchange of various metabolites and a vitamin-derived cofactor.

Abstract A variety of metabolites and a vitamin-derived cofactor are shared between cells coupled by permeable intercellular junctions (gap junctions). The molecular species which pass through the junctions have been identified as 2-deoxy-glucose or 2-deoxy-glucose-6-phosphate, phosphoryl choline or CDP-choline, proline or its precursors and lower glutamated forms of tetrahydrofolate. The active tetraglutamate form of tetrahydrofolate (molecular weight 960) is not shared between coupled cells. These observations support the concept that junctional permeability is selective only with respect to molecular size and that populations of coupled cells will become syncytial with respect to their low molecular weight cytoplasmic components.

Structure of the ductin channel.

Gap junctions appear to be essential components of metazoan animals providing a means of direct means of communication between neighboring cells. They are sieve-like structures which allow cell–cell movement of cytosolic solutes below 1000 MW. The major role of gap junctions would appear to be homeostatic giving rise to groups of cells which act as functional units. Ductin is the major core component of gap junctions and recent structural data shows it to be a four alpha-helical bundle which fits particularly well into a low resolution model of the gap junction channel. Ductin is also the main membrane component of the vacuolar H+-ATPase that is found in all eukaryotes and it seems likely that the gap junction channel first evolved as a housing for the rotating spindle of these proton pumps. Because ductin protrudes little from the membrane, other proteins are required to bring cell surfaces close enough together to form gap junctions. Such proteins may include connexins, a large family of proteins found in vertebrates.

Connexins and the vacuolar proteolipid-like 16-kDa protein are not directly associated with each other but may be components of similar or the same gap junctional complexes☆

Gap junction preparations made from mouse liver plasma membranes by alkali extraction contain variable proportions of connexins (Cx32 and Cx26) and the 16-kDa protein which is closely related or may be identical to the 16-kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and the mediatophore complex. The absence of a stoichiometric relationship suggests that connexins and the 16-kDa protein are not subunits of the same channel complex, but analysis of alkali preparations by isopycnic centrifugation shows both types of protein are in membrane structures of the same buoyant density. Electron microscopic analysis of alkali preparations shows a homogeneous population of gap junctions of uniform morphology and width, suggesting the proteins are in the same or similar structures. The structures containing connexins and the 16-kDa protein can be separated by treatment of the plasma membranes with Triton X-100. After such treatment, the connexins remain associated with dense cellular or extracellular material and the gap junctional structures, after further extraction with N-lauroyl sarcosine and urea, contain only the 16-kDa protein. These detergent-extracted gap junctions are thinner (14.1 nm) than those in alkali preparations (18.4 nm).

A Structural Analysis of the Gap Junctional Channel and the 16K Protein

Membrane proteins have been the focus of much attention in recent years. Advances in the handling of membrane proteins, in protein sequencing technology and in molecular biology have been coupled with high resolution imaging techniques and chemical labelling studies. This has resulted in detailed pictures of a number of membrane proteins including the photoreaction centre (Huber, 1989), acetyl choline receptor (Unwin et al, 1988) and rhodopsin (Findlay & Pappin, 1986). The knowledge which has been gained from these proteins provides a useful framework for the analysis of the organisation of other membrane protein complexes.