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.
Finbow
M E
ME
Beatson Institute for Cancer Research, Wolfson Laboratory for Molecular Pathology, Glasgow, UK.
Shuttleworth
J
J
Hamilton
A E
AE
Pitts
J D
JD
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002500:Centrifugation, Isopycnic; D017630:Connexins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006224:Cricetinae; D003432:Cross-Linking Reagents; D017629:Gap Junctions / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D005976:Glutaral; D008099:Liver / Q000737:chemistry / Q000648:ultrastructure; D008115:Liver Regeneration; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D051381:Rats; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017630:Connexins; D003432:Cross-Linking Reagents; D012967:Sodium Dodecyl Sulfate; D013755:Tetradecanoylphorbol Acetate; D005976:Glutaral