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Preliminary evidence that Clostridium perfringens type A enterotoxin is present in a 160,000-Mr complex in mammalian
1Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Abstract:
Clostridium perfringens type A 125I-enterotoxin (125I-CPE) was bound to rabbit intestinal brush border membranes (BBMs) or Vero cells and then solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS). Solubilized radioactivity was analyzed by gel filtration chromatography on a Sepharose 4B column or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without sample boiling and autoradiography. Specifically bound 125I-CPE extracted from either BBMs or Vero cells was primarily associated with a complex of approximately 160,000 Mr. The CPE complex was partially purified by gel filtration or SDS-PAGE without sample boiling. SDS-PAGE analysis with sample boiling of the partially purified 125I-CPE complex from Vero cells or BBMs suggested that CPE complex contains both a 50,000-Mr protein and a 70,000-Mr protein in approximately equimolar amounts. This result is supported by affinity chromatography with CPE immobilized on Sepharose 4B, which showed the specific interaction of similar size proteins with CPE. The simplest explanation for these results is that CPE (Mr 35,000) interacts with 50,000-Mr and 70,000-Mr eucaryotic proteins to form a membrane-dependent complex of approximately 160,000 Mr. These results suggest that the receptor or target site(s) or both for CPE are similar in both BBMs and Vero cells. The significance of these findings in terms of CPE binding, insertion, and biologic action is discussed.
Insights
Clostridium perfringens enterotoxin (CPE) binds to rabbit intestinal membranes and Vero cells, forming a large complex. This complex involves CPE interacting with host proteins, suggesting conserved receptor sites.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Clostridium perfringens type A produces enterotoxin (CPE), a key virulence factor.
- Understanding CPE's interaction with host cells is crucial for elucidating its mechanism of action.
Purpose of the Study:
- To investigate the molecular interactions of 125I-labeled CPE with rabbit intestinal brush border membranes (BBMs) and Vero cells.
- To identify the host proteins involved in CPE binding and complex formation.
Main Methods:
- Binding of 125I-CPE to BBMs and Vero cells, followed by solubilization with CHAPS.
- Analysis of solubilized complexes using gel filtration chromatography (Sepharose 4B) and SDS-PAGE without boiling.
- Affinity chromatography using immobilized CPE.
Main Results:
- Specifically bound 125I-CPE formed a complex of approximately 160,000 Mr with both BBMs and Vero cells.
- SDS-PAGE analysis indicated the complex contains 50,000-Mr and 70,000-Mr proteins in equimolar amounts.
- CPE (35,000 Mr) likely interacts with these host proteins to form the larger complex.
Conclusions:
- CPE forms a membrane-dependent complex with host proteins of approximately 160,000 Mr.
- The receptor or target sites for CPE appear similar in rabbit BBMs and Vero cells.
- These findings provide insights into CPE binding, insertion, and biological activity.