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Analysis of vertebrate gap junction protein.
Author(s) -
Finbow M.E.,
Shuttleworth J.,
Hamilton A.E.,
Pitts J.D.
Publication year - 1983
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1983.tb01611.x
Subject(s) - biology , vertebrate , evolutionary biology , computational biology , genetics , microbiology and biotechnology , gene
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.