Kinetics of the inhibition of proteinase 3 by elafin.
Excessive proteolytic activity of proteinase 3 (Pr3) has been suggested to be a factor contributing to the pathogenesis of emphysema and other inflammatory disorders. We report here on the kinetics of inhibition of Pr3 by one of its major endogenous inhibitors, the 6-kD inhibitory domain of elafin. The results are consistent with a reaction mechanism in which a single elafin molecule binds a single Pr3 molecule to form a fully reversible complex. The association and dissociation rate constants, and the inhibition constant were measured to be 4.0 x 10(6) M(-1) s(-1), 1.7 x 10(-3) s(-1), and 4.2 x 10(-10) M, respectively. Triton X-100 and dimethyl sulfoxide, which are frequently added in assay mixtures for enzymatic analysis of Pr3 activity, significantly reduced the association rate. A fraction of the total neutrophil content of Pr3 has been reported to be bound to the surface of the plasma membrane of activated and nonactivated neutrophils. In this study, we also measured the reaction rate constants of elafin with Pr3 that had been previously allowed to associate with phospholipid bilayer vesicles. Binding to the model membranes slowed down the association rate to 3.3 x 10(5) M(-1) s(-1), but the membrane-bound Pr3 and elafin formed a more stable complex, with a dissociation rate constant of 9.1 x 10(-4) s(-1). Based on the kinetic parameters determined here and the estimated elafin concentrations in vivo, it may be concluded that elafin plays a limited role in the regulation of proteolytic activity of Pr3 in lung secretions.
Ying
Q L
QL
Departments of Pathology and Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794, USA. qying@path.som.sunysb.edu
Simon
S R
SR
D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000737:chemistry / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008956:Models, Chemical; D053146:Myeloblastin; D017830:Octoxynol / Q000737:chemistry / Q000494:pharmacology; D010743:Phospholipids / Q000737:chemistry; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry* / Q000494:pharmacology; D011656:Pulmonary Emphysema / Q000201:enzymology; D012697:Serine Endopeptidases / Q000737:chemistry*
D003902:Detergents; D004791:Enzyme Inhibitors; D008081:Liposomes; D010743:Phospholipids; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D017830:Octoxynol; D012697:Serine Endopeptidases; D053146:Myeloblastin; D004121:Dimethyl Sulfoxide