Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach.
A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.
Baici
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Department of Rheumatology, University Hospital, Zurich, Switzerland.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004549:Elastin / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012995:Solubility; D013552:Swine
D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine; D004549:Elastin; D010196:Pancreatic Elastase