Proteolysis of cardiac gap junctions during their isolation from rat hearts.
Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfluoride (PMSF) contain a Mr 44,000 to 47,000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a Mr 29,500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. Page Am. J. Physiol. 246:H865-H875, 1984). Rat liver GJ isolated with or without PMSF contain a Mr 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the Mr 44,000 to 47,000 cardiac GJ polypeptide is the precursor of the Mr 29,500 subunit. We show that the Mr 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6 M KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and that in vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure; D005260:Female; D007365:Intercellular Junctions / Q000648:ultrastructure*; D008099:Liver / Q000648:ultrastructure; D008297:Male; D008407:Mast Cells / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D009206:Myocardium / Q000648:ultrastructure*; D009928:Organ Specificity; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D008565:Membrane Proteins; D010664:Phenylmethylsulfonyl Fluoride