Sodium dodecyl sulfate-stable complexes between serpins and active or inactive proteinases contain the region COOH-terminal to the reactive site loop.
Recently inhibitors of the serpin family were shown to form complexes with dichloroisocoumarine (DCI)-inactivated proteinases under native conditions (Enghild, J. J., Valnickova, Z., Thøgersen I., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 20159-20166). This study demonstrates that serpin-DCI/proteinase complexes resist dissociation when analyzed in reduced SDS-polyacrylamide gel electrophoresis. Previously, SDS-stable serpin-proteinase complexes have been observed only between serpins and catalytically active proteinases. The stability of these complexes is believed to result from an acyl-ester bond between the active site Ser195 of the proteinase and the alpha-carbonyl group of the scissile bond in the reactive site loop. We have further analyzed the structure of the SDS-stable serpin-proteinase and serpin-DCI/proteinase complexes. The results of these studies demonstrate the presence of the COOH-terminal region of the serpin in both complexes. Since (i) modification of Ser195 does not prevent formation of SDS-stable complexes and (ii) COOH-terminal peptides are present in both complexes, the previously described mechanism does not sufficiently explain the formation of SDS-stable complexes.
Christensen
S
S
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Valnickova
Z
Z
Thøgersen
I B
IB
Pizzo
S V
SV
Nielsen
H R
HR
Roepstorff
P
P
Enghild
J J
JJ
D000818:Animals; D001665:Binding Sites; D003374:Coumarins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D049934:Isocoumarins; D007700:Kinetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012694:Serine; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D003374:Coumarins; D049934:Isocoumarins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D012967:Sodium Dodecyl Sulfate; D012694:Serine; C046235:3,4-dichloroisocoumarin; D014357:Trypsin