Effect of polynucleotides on the inhibition of neutrophil elastase by mucus proteinase inhibitor and alpha 1-proteinase inhibitor.
DNA released from neutrophils at sites of inflammation may modulate tissue proteolysis. We used tRNA and synthetic polynucleotides as models of DNA to study the influence of polynucleotides on the inhibition of neutrophil elastase by its endogenous inhibitors alpha1-proteinase inhibitor (alpha1-PI) and mucus proteinase inhibitor (MPI). Affinity chromatography showed that polynucleotides form electrostatic complexes with elastase and MPI but not with alpha1-PI, the highest affinity being for MPI. The tight-binding partial inhibition of elastase by polynucleotides was used to calculate the Kd of the elastase-polynucleotide complexes which ranged from 4 microM to 21 nM. One mole of tRNA was able to bind 9 mol of elastase. Polydeoxycytosine and tRNA significantly impaired the reversible inhibition of elastase by MPI: they moderately increased the rate of enzyme-inhibitor association, strongly enhanced the rate of complex dissociation, and lowered the enzyme-inhibitor affinity by factors of 34 and 134, respectively. The two polynucleotides also decreased the rate of the irreversible inhibition of elastase by alpha1-PI by factors of 30 and 3, respectively. Polynucleotides also changed the mechanism of inhibition of elastase by the two inhibitors from a one-step inhibition reaction to a two-step binding mechanism. Our data may help explain why proteolysis may occur at sites of inflammation despite the presence of active proteinase inhibitors.
Belorgey
D
D
Laboratoire d'Enzymologie, INSERM Unité 392, Université Louis Pasteur de Strasbourg, Illkirch, France.
Bieth
J G
JG
D001665:Binding Sites; D002846:Chromatography, Affinity; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D046911:Macromolecular Substances; D011089:Polydeoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology*; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000378:metabolism / Q000502:physiology*; D012343:RNA, Transfer / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000378:metabolism / Q000502:physiology*; D000515:alpha 1-Antitrypsin / Q000378:metabolism / Q000502:physiology*
D046911:Macromolecular Substances; D011089:Polydeoxyribonucleotides; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D012343:RNA, Transfer; D019272:Leukocyte Elastase