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Characterization of a membrane-bound arginine-specific serine protease from rat intestinal mucosa
K Kishi1, K Yamazaki, I Yasuda
1School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Abstract:
Previously we isolated and characterized a membrane-bound, arginine-specific serine protease from pig intestinal mucosa [J. Biol. Chem. 269, 32985-32991 (1994)]. For further characterization of this type of enzyme, we cloned a cDNA from rat intestinal mucosa encoding the precursor of a similar protease. The partial amino acid sequences determined for the pig enzyme were found to be shared almost completely by the rat enzyme. The serine protease domain of the rat enzyme, heterologously expressed in Escherichia coli, specifically cleaved Arg (or Lys)-X bonds with a marked preference for Arg-Arg or Arg-Lys, similar to the pig enzyme. The mRNA for the rat enzyme was shown to be distributed mainly in intestine, and the enzyme was detected in the duodenal mucosa as a 70 kDa protein. Immunohistochemical analysis of the small intestinal tissue showed that the enzyme is localized mainly on brushborder membranes.
Insights
Researchers cloned a rat intestinal protease, similar to a pig enzyme, that cleaves specific arginine bonds. This serine protease is primarily found on the brush border membranes of the small intestine.
Area of Science:
- Biochemistry
- Molecular Biology
- Gastroenterology
Background:
- A membrane-bound, arginine-specific serine protease was previously isolated from pig intestinal mucosa.
- Further characterization of this enzyme type is needed.
Purpose of the Study:
- To clone and characterize a cDNA encoding a protease similar to the pig enzyme from rat intestinal mucosa.
- To investigate the substrate specificity and localization of the rat enzyme.
Main Methods:
- cDNA cloning from rat intestinal mucosa.
- Heterologous expression of the serine protease domain in Escherichia coli.
- Enzyme activity assays to determine substrate specificity.
- mRNA distribution analysis using Northern blotting.
- Immunohistochemical analysis of rat small intestinal tissue.
Main Results:
- A cDNA encoding a precursor of a protease similar to the pig enzyme was cloned from rat intestinal mucosa.
- The rat enzyme shares significant sequence homology with the pig enzyme.
- The expressed serine protease domain specifically cleaved Arg(or Lys)-X bonds, with a preference for Arg-Arg or Arg-Lys.
- The enzyme's mRNA was predominantly found in the intestine.
- The 70 kDa protein was detected in the duodenal mucosa and localized to brush-border membranes.
Conclusions:
- The rat intestinal enzyme is a serine protease with similar substrate specificity to the previously characterized pig enzyme.
- The enzyme is primarily expressed in the intestine and localized to the brush-border membrane.
- This study provides insights into the structure, function, and localization of intestinal serine proteases.