Immunoblot analysis of Met-α₂AP cleaving activity of APCE in pooled fractions from each chromatographic step. (A) APCE activity increased at each successive purification step. P, Q, T, or I represents APCE activity of each chromatographic pool of samples with Z-Gly-Pro-AMC cleaving activity. The total protein used from each chromatographic step was as follows: P, 54 μg; Q, 9 μg; T, 0.6 μg; or I, 0.2 μg. Met-α₂AP cleavage was demonstrated by immunoblot analysis using an antibody specific for its N-terminal peptide. (B) Incubation of Met-α₂AP with the APCE activity pool from immunoaffinity chromatography (0.2 μg) diminished in a time-dependent manner when assessed by an antibody specific for the N-terminal peptide of Met-α₂AP (left panel), but did not diminish α₂AP band intensity or generate bands less than 70-kDa when assessed by a monoclonal antibody to the C-terminal region of Met-α₂AP or Asn-α₂AP (right panel). (C) APCE (amount shown above each gel lane) concentration-dependent cleavage of Met-α₂AP (5 μg) detected by an antibody specific for Met-α₂AP.