Related Experiment Videos
Proteolysis of cardiac gap junctions during their isolation from rat hearts
Abstract:
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.
Insights
Heart gap junctions (GJ) degrade due to a serine protease released from mast cells during isolation. This protease, inhibited by PMSF, differs from liver GJ composition, highlighting tissue-specific differences in gap junction protein structure.
Area of Science:
- Cellular Biology
- Biochemistry
- Proteomics
Background:
- Gap junctions (GJ) are crucial for intercellular communication.
- Cardiac GJ proteolysis during isolation yields a smaller polypeptide, unlike liver GJ.
- The origin and nature of the cardiac protease remain unclear.
Purpose of the Study:
- Investigate the cardiac protease responsible for GJ breakdown.
- Determine why liver GJ are resistant to similar proteolysis.
- Clarify the relationship between cardiac GJ polypeptide forms.
Main Methods:
- Isolation of rat heart and liver gap junctions with and without protease inhibitors.
- Protease activity assay using phenylmethylsulfonylfluoride (PMSF), soybean trypsin inhibitor, and chymostatin.
- In vivo mast cell degranulation using compound 48/80.
Main Results:
- Cardiac GJ contain a Mr 44,000-47,000 polypeptide, the unproteolyzed connexon subunit.
- Proteolysis occurs in high ionic strength solutions (0.6 M KI), releasing serine protease from mast cells.
- The protease is sensitive to PMSF and partially inhibited by soybean trypsin inhibitor and chymostatin.
- In vivo mast cell degranulation did not prevent cardiac GJ breakdown during isolation.
Conclusions:
- Cardiac GJ proteolysis is mediated by a mast cell-derived serine protease.
- This protease is released under specific isolation conditions (high ionic strength).
- Rat heart and liver GJ exhibit distinct protein compositions and susceptibility to proteolysis.