TABLE 3.
Metal ion and inhibitor effects on the activity of the purified protease
| Compound (mM concn) | Caseinolytic activity (%)a |
|---|---|
| None | 100 |
| CaCl2 (1) | 124.2 ± 8 |
| CaCl2 (10) | 132.1 ± 10 |
| MgCl2 (1) | 122.1 ± 9 |
| MgCl2 (10) | 158.4 ± 6 |
| ZnCl2 (1) | 84.13 ± 7 |
| ZnCl2 (10) | 10.5 ± 3 |
| MnCl2 (1) | 94.3 ± 4 |
| MnCl2 (10) | 66.6 ± 3 |
| Phenylmethysulfonyl fluoride (1) | 100 ± 7 |
| 1,10-Phenanthroline (1) | 5.5 ± 1 |
| Dithiothreitol (1) | 28.4 ± 3 |
| EGTA (1) | 49.6 ± 4 |
| EDTA (1) | 6.6 ± 3 |
| EDTAb (5) | 5.1 ± 2 |
| EDTA (5) + CaCl2 (10) | 43.6 ± 6 |
| EDTA (5) + MgCl2 (10) | 55.7 ± 5 |
| EDTA (5) + CaCl2 (5) + MgCl2 (5) | 57.4 ± 6 |
Caseinolytic activity is expressed as the percentage of the control value (with no addition). The values are the averages ± standard deviations for two independent experiments. Purified protease (∼1.7 μg) was incubated in Tris buffer containing each cation. For inhibition studies the enzyme (∼1.7 μg) was preincubated with the inhibitor compound for 10 min at room temperature, and then caseinolytic activities were determined.
The effect of Ca2+ and Mg2+ on EDTA-inhibited protease was studied by incubating approximately 1.7 μg of enzyme in Tris buffer containing 5 mM EDTA for 10 min at room temperature. The reaction mixture was then made 10 mM with either CaCl2 or MgCl2 or with 5 mM concentrations of each of the two combined. Caseinolytic activity was then determined by incubation for 30 min at 30°C in the presence of 1% azocasein as the substrate.