Accelerated binding of secretory leukoprotease inhibitor to human leukocyte elastase mediated by single-stranded sites in DNA from tracheobronchial mucus.
We have found that preparations of DNA isolated from purulent sputum possess a novel activity which accelerates and stabilizes the binding of human leukocyte elastase to secretory leukoprotease inhibitor, a major endogenous antielastase in the respiratory tract. DNA in sputum is derived from the nuclear debris of disintegrated inflammatory leukocytes, and can attain concentrations ranging from 10(2) to 10(4) micrograms/ml, depending on the severity of pulmonary infection and inflammation. In the presence of 23 micrograms/ml DNA, a concentration lower than those found in most purulent sputa, the rate constant for association of secretory leukoprotease inhibitor with elastase is increased to 1.1 x 10(8) M-1s-1, 44-fold greater than that in the absence of DNA. The equilibrium dissociation constant for the enzyme-inhibitor complex drops to 0.7 pM, two orders of magnitude lower than that in the absence of DNA. The accelerating effect of DNA is further increased by thermal denaturation or by modification with exonuclease III, while it is significantly reduced by digestion with S1 nuclease or by binding of Escherichia coli single-stranded DNA binding protein. The results from these experiments indicate that the structural elements in sputum DNA that are responsible for the accelerating effect have the characteristics of single-stranded sites. Similar kinetic effects on elastase inhibition were also observed with human placental DNA and genomic DNAs from a variety of other species. These findings suggest that DNA in pulmonary secretions may participate in antielastase defense by promoting the binding of secretory leukoprotease inhibitor to leukocyte elastase. The results may have important implications for use of nuclease preparations in mucolytic therapy for cystic fibrosis.
Ying
Q L
QL
Department of Pathology, State University of New York at Stony Brook 11794, USA.
Simon
S R
SR
D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D001980:Bronchi / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism; D009092:Mucous Membrane / Q000378:metabolism; D009093:Mucus / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism*; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000494:pharmacology; D013183:Sputum / Q000378:metabolism; D013696:Temperature; D014132:Trachea / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase