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Identification of an elastase-like activity, that decreased during the differentiation of MEL cells, as a prolyl
T Tsukahara1, S Ishiura, H Sugita
1National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
Abstract:
1. A Suc-APA-MCA hydrolytic activity was significantly decreased in murine erythroleukemia cells during DMSO-induced differentiation, but not in DMSO-resistant cells. 2. The Suc-APA-MCA hydrolytic enzyme was purified by ion exchange, adsorption, gel filtration and affinity chromotographies. The results of the chromatographies showed that only one enzyme hydrolyzed Suc-APA-MCA in MEL cells. 3. This enzyme is more sensitive to hydrolysis by Suc-GPLGP-MCA than Suc-APA-MCA at slightly acidic pH, and its activity is stimulated by 2-mercaptoethanol. 4. A cysteine proteinase inhibitor did not affect the activity, but a specific inhibitor of prolyl endopeptidase, Z-thioprothiazolidine, completely inhibited it. These results suggest that the Suc-APA-MCA hydrolytic enzyme is identical to a prolyl endopeptidase.
Insights
Murine erythroleukemia cell differentiation involves decreased Suc-APA-MCA hydrolytic activity. This activity is linked to a specific enzyme identified as prolyl endopeptidase.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Murine erythroleukemia (MEL) cells are a model for studying cellular differentiation.
- Understanding enzyme activity during differentiation is crucial for cell biology research.
Purpose of the Study:
- To investigate the role of Suc-APA-MCA hydrolytic activity in DMSO-induced differentiation of MEL cells.
- To identify and characterize the enzyme responsible for Suc-APA-MCA hydrolysis in MEL cells.
Main Methods:
- Enzyme purification using multiple chromatography techniques (ion exchange, adsorption, gel filtration, affinity).
- Enzyme activity assays with different substrates and inhibitors.
- Comparison of enzyme activity in differentiating and DMSO-resistant MEL cells.
Main Results:
- Suc-APA-MCA hydrolytic activity significantly decreased in differentiating MEL cells but not in resistant cells.
- A single enzyme was purified and found to hydrolyze Suc-APA-MCA.
- The enzyme exhibited higher sensitivity to Suc-GPLGP-MCA at acidic pH and was stimulated by 2-mercaptoethanol.
- Activity was unaffected by cysteine proteinase inhibitors but completely inhibited by a prolyl endopeptidase specific inhibitor.
Conclusions:
- The Suc-APA-MCA hydrolytic enzyme in MEL cells is identified as prolyl endopeptidase.
- This enzyme's activity is modulated during DMSO-induced cellular differentiation.