A COMPARISON OF THE INHIBITION OF PORCINE PANCREATIC ELASTASE AND HUMAN NEUTROPHIL ELASTASE BY ALPHA-CRYSTALLIN

Bovine lens alpha-crystallin inhibited both porcine pancreatic elastas e (PPE) and human neutrophil elastase (HNE), but not in the same manne r. PPE was immediately inhibited with a stoichiometry of 10 moles of P PE inhibited per mole of alpha-crystallin. The inhibition was markedly decreased by the addition of even low levels of salts. The inhibition was transient, as PPE activity returned to normal with a t(1/2) of 30 min even in low salt. HNE required a short preincubation to show maxi mum inhibition with a stoichiometry of approximately one mole of HNE i nhibited per mole of alpha-crystallin. The inhibition of HNE was only slightly decreased by the addition of 0.1 M salt, and HNE activity ret urned slowly exhibiting a tin of 30 hrs under these conditions. The in hibition of each enzyme by alpha-crystallin was evaluated by Dixon plo ts giving K-i values of 1.5 nM for PPE and 0.25 nM for HNE. DFP-trypsi n was able to compete with PPE for binding to alpha-crystallin and cau se the release of PPE already bound to alpha-crystallin. The inhibitio n of HNE, however, was unaffected by the addition of DFP-trypsin. A mi xture of HNE and alpha-crystallin in 0.1 M NaCl was incubated at 25 de grees C for 6 hours. Aliquots showed a slow, continuous cleavage of th e or-crystallin subunits by SDS-PAGE, but little or no increase in HNE activity. A similar experiment with PPE in 0.1 M NaCl showed no inhib ition and a significant cleavage of alpha-crystallin after only one mi nute of incubation. These data argue for distinct inhibitory mechanism s and binding sites for these two elastase enzymes.