SOURCE. Murine macrophages.
Murine Metallo-elastase also know as murine macrophage elastase (MME) is a zinc-containing metalloproteinase that hydrolyzes insoluble.
The 53 Kd protein is prepared as a proenzyme that can be activated by trypsin like proteases or p-aminophenylmercuric acetate (APMA) the 22 Kd active metalloproteinase. This product is available in the proenzyme form.
Purity is greater than 95% by SDS-PAGE.
chromatographically purified, lyophilized, water soluble powder.
Store at -20°C. Will remain stable for 6 to 8 months at -20°C.
MW (activated) = 22,500
MW (non-activated) = 52,000
75 units per mg protein.
one unit will degrade 1µgm of insoluble elastin per hour at 37°C in 25 mM Tris pH 7.8 containing 10 mM CaCl2 and 150 mM NaCl. Must be activated with trypsin or APMA.
Prepared by the modifications of the method of Banda and Werb (60, 61).
Use as buffer 25 mM Tris-HCl pH 7.8 containing 10 mM CaCl2 and 150 mM NaCl.
Pancreatic trypsin, 20 µg/ml in buffer or p-aminophenylmercuric acetate (APMA), 12 mg/ml in buffer.
To 50µg elastase in 100 µl buffer, add 5 µl trypsin and incubate at 25°C for 30 minutes. Trypsin may then be inhibited by adding 1 µg SBTI in buffer.
To 50 µg elastase in 100 µl buffer, add 5 µl APMA and incubate at room temperature for 4 hours.
Procedure for determination of elastolytic activity by spectrophotometry.
Preparation of substrate:
Insoluble bovine neck ligament elastin (EPC No. ES70, pass 400 Mesh) is suspended (20mg/ml) in 0.025 M HEPES, 0.150 M NaCl and 10 mM CaCl2 containing 0.01% Trixton X-100, pH 7.8. The suspension is stirred for 18 hours at room temperature. The suspension is then washed over a fritted glass filter and resuspended at a final concentration of 10 mg/ml in the HEPES buffer.
Aliquots of substrate and MME are pipetted in 20 ml flasks, the flasks are stoppered and incubated at 37°C in a shaking water bath. At various times, including time '0', 1 ml samples are removed into centrifuge tubes containing 0.5 ml 0.1 M acetic acid, 4.0 M NaCl, pH 4.7, to stop the reaaction. Following centrifugation at 10,000 x g for 10 minutes, the soluble peptides are determined at 280 nm.