TABLE 3.
Effects of various compounds on the peptide hydrolyzing activities of the 20S proteasome from M. thermophila
| Compound | Concn (mM) | Activitya
|
|
|---|---|---|---|
| CL | PG | ||
| None | 100 ± 0.5 | 100 ± 0.2 | |
| ATP | 1 | 100 ± 1.1 | 131 ± 2.1 |
| GTP | 1 | 100 ± 1.0 | 98 ± 2.0 |
| MgCl2 | 10 | 151 ± 9.8 | 54 ± 1.0 |
| MnCl2 | 10 | 129 ± 2.2 | 51 ± 1.2 |
| ZnCl2 | 10 | 140 ± 5.6 | 3 ± 0.5 |
| CaCl2 | 100 | 204 ± 3.0 | 45 ± 1.0 |
| KCl | 300 | 177 ± 6.0 | 33 ± 0.5 |
| NaCl | 100 | 150 ± 9.5 | 99 ± 1.0 |
| SDS | 0.7 | 208 ± 14 | 37 ± 0.5 |
| Spermidine | 1 | 103 ± 7.9 | 94 ± 4.0 |
| EDTA | 5 | 98 ± 3.8 | 103 ± 2.1 |
| Cysteine | 1 | 100 ± 2.6 | 96 ± 3.0 |
| 3,4-Dichloroisocoumarin | 0.022 | 0 ± 0.1 | 3 ± 0.5 |
| Ac-Leu-Leu-Nle-alb | 0.13 | 7 ± 1.0 | 26 ± 2.0 |
a
Expressed as relative activity, where activity with no added effector molecule is 100%. CL activity was assayed by using 20 μM Suc-LLVY-Amc, where 100% specific activity was 0.7 nmol of product/min/mg. PG activity was measured by using 250 μM Cbz-LLE-βNa, where 100% specific activity was 12.0 nmol of product/min/mg. The 20S proteasome used was produced in E. coli. The enzyme was preincubated with the indicated compound in 20 mM Tris-HCl (pH 7.2) at 21°C for 10 min prior to assay at 37°C. The final concentration of the 20S proteasome in the assay was 0.01 mg/ml.
b
Ac-Leu-Leu-Nle-al; acetyl-Leu-Leu-norleucine-al.