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Soluble metalloendopeptidase (MMP-7ase) activity in mouse kidney cytosol
1Department of Biochemistry, Virginia Polytechnic and State University, Blacksburg 24061.
Abstract:
Previously, MMP-7ases were isolated from rat skeletal muscle by gel filtration and anion exchange chromatography. The enzyme that hydrolyzed succinyl-Ala-Ala-Pro-Phe-AMC (AMC: 7-amino-4-methyl-coumarin) was inhibited by EDTA. In this study we attempted to isolate MMP-7ase from mouse kidney. The isolation procedure was the same as that previously used for skeletal muscle. Kidneys of ICR mice were homogenized and, after centrifugation, the supernatant fraction was subjected to gel filtration chromatography. The fraction with the highest activity (Mr 67-72 kDa) was subjected to anion exchange chromatography, which showed three peaks of activity. The second peak hydrolyzed succ-Ala-Ala-Pro-Phe-AMC, but had low activity against Arg- or Ala-AMC. This peak was a single protein (Mr 68-72 kDa) and its activity could be inhibited with EDTA. Several tri- and tetrapeptide derivatives were tested as substrates for this enzyme and the best was found to be succ-Ala-Ala-Pro-Phe-AMC. We can conclude that mouse kidney cytosol contains a metalloendopeptidase similar to muscle MMP-7ase.
Insights
Researchers isolated a metalloendopeptidase (MMP-7ase) from mouse kidney cytosol. This enzyme is similar to the one previously found in rat skeletal muscle and prefers specific peptide substrates.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Metalloendopeptidases (MMP-7ases) have been previously isolated from rat skeletal muscle.
- These enzymes hydrolyze specific peptide substrates and are inhibited by EDTA.
Purpose of the Study:
- To isolate and characterize MMP-7ase from mouse kidney cytosol.
- To compare the properties of mouse kidney MMP-7ase with the previously identified rat skeletal muscle enzyme.
Main Methods:
- Mouse kidneys were homogenized, and the supernatant was subjected to gel filtration chromatography.
- Active fractions were further purified using anion exchange chromatography.
- Enzyme activity was assessed using various peptide substrates, including succinyl-Ala-Ala-Pro-Phe-AMC.
Main Results:
- A metalloendopeptidase (MMP-7ase) was successfully isolated from mouse kidney cytosol.
- The enzyme exhibited highest activity against succinyl-Ala-Ala-Pro-Phe-AMC and was inhibited by EDTA.
- The molecular weight of the purified enzyme was determined to be 68-72 kDa.
Conclusions:
- Mouse kidney cytosol contains a metalloendopeptidase similar to the MMP-7ase found in rat skeletal muscle.
- The isolated enzyme displays substrate specificity for succinyl-Ala-Ala-Pro-Phe-AMC.
- This finding contributes to understanding the diversity and tissue distribution of metalloendopeptidases.