Table I.

Specific AP and NPA amidase activities of PM proteins at each step of purification

SubstratePMSolubilizedS300Q ResinNPA Peak IINPA Peak IIINPA Peak II + III
nmol min−1 mg−1
Tyr-AFC31.616.678.1226.859.37.7392.4
Ala-Pro-AFC17.613.515.722.489.51.966.3
Trp-AFC20.217.13.34.72.21.74.7
Pro-AFC16.715.627.931.527.15.946.1
Ala-AFC2.62.416.162.827.27.584.6
Leu-AFC10.39.418.219.72.53.12.1
Ala-NA2.11.62.731.632.118.445.7
NPA6.34.45.825.117.19.542.1

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.