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Published on: May 14, 2016
Identification of a proteolytic activity which responds to anticarcinogenic protease inhibitors in C3H-10T1/2 cells
1Harvard School of Public Health, Boston, MA 02115.
Abstract:
Untransformed and malignantly-transformed mouse embryo fibroblasts were found to contain an enzymatic activity which hydrolysed the synthetic substrate, Suc-Ala-Ala-Pro-Phe-AMC. This activity, of approximate molecular weight 55,000, which has been partially purified by ion-exchange and gel-filtration chromatography, was maximally active at neutral pH, associated with subcellular organelles or membranes and inhibited by EDTA, EGTA, phosphoramidon and 1,10 phenanthroline, but not by PMSF or pepstatin, indicating that it may be a metalloprotease. Several other protease inhibitors, such as chymostatin, TPCK and the BBI from soybean, were also potent inhibitors of the activity; these same inhibitors are known to suppress the malignant transformation of mouse embryo fibroblast cells in vitro induced by X-irradiation or chemical carcinogens. In contrast, SBTI and pepstatin, which did not inhibit this activity, also do not suppress malignant transformation. These results suggest that this enzyme may be involved in attainment of the transformed state.
Insights
A novel metalloprotease activity was identified in mouse embryo fibroblasts. This enzyme
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Malignant transformation of cells is a complex process.
- Protease activities are implicated in cellular regulation and disease.
- Understanding enzymes involved in transformation is crucial for cancer research.
Purpose of the Study:
- To identify and characterize enzymatic activities in transformed mouse embryo fibroblasts.
- To investigate the potential role of a specific protease in malignant transformation.
Main Methods:
- Enzyme assays using synthetic substrate Suc-Ala-Ala-Pro-Phe-AMC.
- Partial purification via ion-exchange and gel-filtration chromatography.
- Characterization of enzyme properties including pH optimum, subcellular localization, and inhibitor sensitivity.
Main Results:
- An enzymatic activity (MW ~55,000) was detected in both untransformed and transformed cells.
- The enzyme exhibited neutral pH optimum, membrane association, and inhibition by metalloprotease inhibitors (EDTA, EGTA, phosphoramidon, 1,10 phenanthroline).
- Potent inhibition of this activity by chymostatin, TPCK, and soybean BBI correlated with their known ability to suppress malignant transformation.
Conclusions:
- The identified enzyme is likely a metalloprotease involved in cellular processes.
- The correlation between inhibition of this enzyme and suppression of malignant transformation suggests its involvement in the transformation process.
- This protease may play a role in the attainment of the transformed cellular phenotype.
