CHARACTERIZATION OF HUMAN LEUKOCYTE ELASTASES: INHIBITION BY CHELATORS, ROLE OF ZINC AND CALCIUM AND EFFECT OF CATIONS AND ANIONS ON ACTIVITY (METALLOENZYME, ISOZYMES, HPLC PURIFICATION)

Neutrophil elastases are released during acute and chronic inflamatory states and are able to hydrolyse a variety of protein substrates. Although studies have indicated that Pms-F and Dip-F can inhibit the leukocyte enzymes, some reports indicated that EDTA and EGTA are also effective inhibitors of this enzyme.;At each stage of purification the quantity of Zn increased, reaching a molar ratio of 2:1 with elastase in the most purified samples. Calcium content was selectively elevated during the earlier stages of purification but decreased to a ratio of 0.25 to 1 with elastase at the final HPLC step. Leukocyte elastase could be inhibited by EDTA, EGTA and 1,10 phenanthroline; EGTA was the most efficient inhibitor, EDTA the least. Inhibition was non-competitive and reversible only if the time of preincubation was relatively short, indicating the instability of the apoenzyme. The concentration of chelator required to show significant inhibition of elastase was also dependent upon the stage of purity and the ionic strength of the reaction mixture. Inhibition by EGTA, followed by the removal of EGTA, could be reversed by Zn. In the presence of EGTA the enzyme could be returned to full activity by the addition of Zn, Mn and Ca, but not Mg or Na. Elastase activity could also be modulated by various monovalent and divalent ions. The regulation of leukocyte elastase through the removal of intrinsic catalytic or regulatory metals, or manipulation of the ionic environment, provides a novel approach to the control of this enzyme which suggest possible physiologic applications.;Neutrophil elastases were purified by a three step procedure consisting of one G-75 Sephadex and two HPLC elutions. The elastases cross-reacted with antibodies to human neutrophil elastase. Three bands with molecular weights between 26,000 and 29,000 were observed by gel electrophoresis. With HPLC separation four distinct elastase activities, assayed with H('3)-elastin as substrate, could be demonstrated.