CHARACTERIZATION OF HUMAN AIRWAY EPITHELIAL-CELL LEUKOTRIENE A(4) HYDROLASE

We have previously shown that human airway epithelial cells contain le ukotriene A(4) (LTA(4)) hydrolase activity. To characterize this activ ity further, airway epithelial cells, cultured to confluence, were dis rupted by sonication and were fractionated at 15,000 and 100,000 x g. Enzymatic activity was assessed by incubating fractions with 15 mu M L TA(4) at 37 degrees C for 15 min. LTA(4) hydrolase activity was presen t in the 15,000 x g and the 100,000 x g supernatants and was inactivat ed by heating at 56 degrees C or by pronase, as is the case for neutro phil LTA(4) hydrolase. However, the epithelial cell enzyme had a slowe r time course for product generation and demonstrated a different dose -response relationship to substrate when compared with the neutrophil. Kinetic analysis revealed nonlinear plots for epithelial data, most c onsistent with an enzyme that has multiple active sites. Immunoblottin g, performed with anti-neutrophil LTA(4) hydrolase antibody, recognize d two bands in epithelial cell 15,000 x g supernatant (M(r) of 69,000 and 110,000-115,000). When resolved by gel filtration chromatography, only the M(r) 69,000 protein had enzymatic activity. Anion exchange ch romatography of epithelial cell samples revealed that LTA(4) hydrolase and aminopeptidase activity did not co-elute, whereas one of three pe aks of aminopeptidase activity did co-elute in chromatograms of neutro phil samples. Immunoblots of proteolytic digests of partially purified M(r) 69,000 protein from epithelial cells and neutrophils revealed di fferent immunoreactive bands. The digest of the M(r) 110,000-115,000 p rotein revealed no immunoreactive bands. Repeat kinetic analysis on 17 9-fold purified epithelial LTA(4) hydrolase again revealed that it lac ked significant aminopeptidase activity and retained its unique kineti c properties. These findings confirm that epithelial cells and neutrop hils have structurally and functionally related LTA(4) hydrolase enzym es, but further suggest that these enzymes are not identical.