Elastin-derived peptide binding to a hydrophobic domain on neutrophil elastase.
To understand the contributions of binding of elastin to domains removed from the active site of neutrophil elastase, we isolated an elastin-derived peptide (EDP) fraction, which we have previously shown was tightly linked to neutrophil elastase after prolonged digestion of elastin but which can be released from the enzyme with hydroxylamine. Elastin from human aorta was incubated with human neutrophil elastase under conditions favoring proteolysis. Low molecular weight species, including free EDP, were separated from the protein fraction by a small centrifuged gel filtration column. The high molecular weight protein fraction was subjected directly to 0.5 M hydroxylamine. The reaction mixture was then fractionated on a phosphocellulose column using an ionic gradient. A fraction was collected that exhibited fluorescence with a peak at approximately 410 nm when excited at 320 nm, indicating the presence of desmosine and (or) isodesmosine. A second peak with amidolytic activity towards methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (MeOSucAAPVpNa), but no fluorescence at 410 nm was also detected at the same elution volume where free elastase appeared. After removal of low molecular weight digestion products but prior to treatment with hydroxylamine, the putative elastase-EDP complex possessed no amidolytic activity towards MeOSucAAPVpNa. When the liberated EDP was added to elastase in an amidolytic assay, the EDP behaved as only a partial noncompetitive inhibitor (Vmax/Vmax approximately 90%), but bound with high affinity to neutrophil elastase (Ki congruent to 29 nM), as detected by its ability to quench elastase endogenous fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)
Tyagi
S C
SC
Department of Medicine, University of Missouri, Columbia 65212, USA.
Simon
S R
SR
D001011:Aorta; D003895:Desmosine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004549:Elastin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005450:Fluorescamine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D066298:In Vitro Techniques; D007524:Isodesmosine / Q000378:metabolism; D007700:Kinetics; D019272:Leukocyte Elastase; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D006898:Hydroxylamines; D010446:Peptide Fragments; D003895:Desmosine; D019811:Hydroxylamine; D005450:Fluorescamine; D004549:Elastin; D007524:Isodesmosine; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase