Figure 6.

(A) α₂M capture assay shows that the anti-ST3 antibody blocks ST3 function. Anti-ST3 antibody inhibited the formation of ST3–α₂M complex. In vitro–synthesized, ³⁵S-labeled ST3 catalytic domain was incubated with α₂M (1 μg in 10 μl reaction) in the absence (lanes 1 and 2) or presence of anti-ST3 antiserum (lanes 3–6) or preimmune serum (lanes 7–10). The resulting mixture was electrophoresed on a native polyacrylamide gel. The gel was stained with Coomassie blue (bottom) and then dried and autoradiographed (top). The serum concentrations were 0.5% for lanes 3 and 7, 1% for lanes 4 and 8, and 2% for lanes 5, 6, 9, and 10. Lane 11 had 3.7 μg α₂M to facilitate its identification by Coomassie blue staining. The arrowhead and arrow indicate the position of α₂M and α₂M–ST3 complex, respectively. (α₂M and α₂M–ST3 complex migrated at the same position. This is because α₂M is 725 kD whereas ST3 catalytic domain is only ∼20 kD. The small size difference between α₂M and α₂M–ST3 complex could not be resolved on this gel.) The faster migrating band in lanes 3–6 is the antibody–ST3 complex. The band migrating above the α₂M–ST3 complex is likely due to the nonspecific binding of ST3 by a protein in the in vitro translation extract, as it was also present in the absence of any α₂M (lane 1) and a protein at this position was also detectable by Coomassie blue staining in all lanes, except lane 11, which had only α₂M. Unlike α₂M–ST3, this band was not inhibited by the antibody. Its apparent increase in lanes 3–6 was due to the increased background smear caused by the anti-ST3 antibody. (B) Anti-ST3 antibody prevents cleavage of α1-antitrypsin by ST3. Purified ST3 catalytic domain was incubated with α1-antitrypsin in vitro in the presence of increasing concentrations (1, 2.5, 5, and 10%) of anti-ST3 or preimmune serum. The full-length and large fragment of α1-antitrypsin generated by ST3 cleavage were detected by Western blot analysis of the reaction product. Note that as little as 1% of the antiserum inhibited all of the cleavage of α1-trypsin by ST3 (lane 3) and most of the binding of ST3 to α₂M (A, lane 4). The arrow and arrowhead indicate the position of the ST3-generated large fragment and full-length α1-antitrypsin, respectively.