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Isolation and some properties of a cathepsin E type proteinase from rat spleen
B Wiederanders1, S Schaper, H Kirschke
1Institute of Biochemistry, Faculty of Medicine, Martin-Luther-University Halle/Wittenberg.
Abstract:
Cathepsin E (EC 3.4.23.--) has been isolated from rat spleen. The procedure included autolysis at pH 4.2 which was probably the reason why we isolated a polypeptide of Mr 42 kDa instead of 90 kDa. The latter is reported in the literature to be the Mr of native cathepsin E. The enzyme dissociates under reducing conditions in two identical monomers. In our preparation a mechanism different from reduction must be active producing the 42 kDa polypeptide. This enzyme was hard to distinguish from cathepsin D (EC 3.4.23.5.) which shows similar properties such as size, substrate specificity, stability in 6 M urea, and dependence of the activity on pH. The clear distinction between the two enzymes was proven on the basis of immunochemical reactions. Antibodies to both cathepsins, D and E, did not show any crossreaction with the nonrelated antigen.
Insights
Researchers isolated rat spleen Cathepsin E, yielding a 42 kDa polypeptide due to autolysis, distinct from the native 90 kDa form. Immunochemical analysis confirmed its unique identity, differentiating it from Cathepsin D.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Cathepsin E is an aspartic protease with known roles in protein degradation.
- The native molecular weight of Cathepsin E is reported as 90 kDa.
- Distinguishing Cathepsin E from Cathepsin D is challenging due to similar biochemical properties.
Purpose of the Study:
- To isolate and characterize Cathepsin E from rat spleen.
- To investigate the discrepancy in molecular weight observed during isolation.
- To establish clear differentiation between Cathepsin E and Cathepsin D.
Main Methods:
- Isolation of Cathepsin E from rat spleen.
- Enzyme characterization including molecular weight determination.
- Autolysis at pH 4.2.
- Immunochemical analysis using specific antibodies against Cathepsin D and E.
Main Results:
- A 42 kDa polypeptide of Cathepsin E was isolated, differing from the native 90 kDa form, likely due to autolysis.
- The isolated enzyme showed properties similar to Cathepsin D, including size, substrate specificity, and pH dependence.
- Immunochemical assays confirmed no cross-reactivity, definitively distinguishing the isolated Cathepsin E from Cathepsin D.
Conclusions:
- Rat spleen Cathepsin E can be isolated as a 42 kDa form under specific autolytic conditions.
- Autolysis, not reduction, appears responsible for generating the smaller polypeptide.
- Immunochemical methods provide a reliable means to distinguish Cathepsin E from Cathepsin D.