Time kinetics of proteolytic processing of PBP and CTAP-III on coincubation with PMN.

Neutrophils were coincubated with 500 nmol/L of either NAP-2 precursor for the time period indicated, and supernatants (SN) were further analyzed. (A) Western blot analysis of SN. 10 μL of each SN was separated by SDS-PAGE, blotted, and immunostained with either antiserum Rα-βTG (binding to all known β-TG Ag isoforms) or Rα-70/2 (detecting the C-terminal residue in β-TG-Ag). 50 ng NAP-2 was run in parallel for comparison. Data from 1 of 3 representative experiments are shown. N indicates NAP-2; P, PBP; C, CTAP-III. (B) Determination of NAP-2 activity equivalents in SN. The potential NAP-2 concentrations in the SN were determined by comparison with purified NAP-2 in the degranulation assay as outlined in “Materials and methods.” Shown are means ± SD of data obtained in 3 independent experiments. The initial velocity, V, was defined as the increase in NAP-2 concentration per minute on the basis of the first time point at which significantly elevated NAP-2 levels could be detected. This time point differed between the precursors (C, 30 minutes; P, 10 minutes). ●, CTAP-III (500 nmol/L); ▴, PBP, (500 nmol/L).