Specific AP and NPA amidase activities of PM proteins at each step of purification
| Substrate | PM | Solubilized | S300 | Q Resin | NPA Peak II | NPA Peak III | NPA Peak II + III |
|---|---|---|---|---|---|---|---|
| nmol min−1 mg−1 | |||||||
| Tyr-AFC | 31.6 | 16.6 | 78.1 | 226.8 | 59.3 | 7.7 | 392.4 |
| Ala-Pro-AFC | 17.6 | 13.5 | 15.7 | 22.4 | 89.5 | 1.9 | 66.3 |
| Trp-AFC | 20.2 | 17.1 | 3.3 | 4.7 | 2.2 | 1.7 | 4.7 |
| Pro-AFC | 16.7 | 15.6 | 27.9 | 31.5 | 27.1 | 5.9 | 46.1 |
| Ala-AFC | 2.6 | 2.4 | 16.1 | 62.8 | 27.2 | 7.5 | 84.6 |
| Leu-AFC | 10.3 | 9.4 | 18.2 | 19.7 | 2.5 | 3.1 | 2.1 |
| Ala-NA | 2.1 | 1.6 | 2.7 | 31.6 | 32.1 | 18.4 | 45.7 |
| NPA | 6.3 | 4.4 | 5.8 | 25.1 | 17.1 | 9.5 | 42.1 |
Fractions (100 ng) from each purification step were assayed for activity after dialysis against AP assay buffer for 1 h at 4°C. AFC conjugate concentrations were 20 μm. Ala-β-naphthylamide (NA) and NPA concentrations were 100 μm. PM, PM-enriched membranes; Solubilized, detergent-solubilized PM proteins; S300, Sephacryl S-300 PMSF-insensitive peak fraction; Q resin, Q anion-exchange PMSF-insensitive peak fraction; NPA peaks I through III, fractions (or combinations of fractions) corresponding to NPA affinityA 280 peaks (Fig. 1D). All assays were repeated at least three times with similar results.