Characterization of human airway epithelial cell leukotriene A4 hydrolase.
We have previously shown that human airway epithelial cells contain leukotriene A4 (LTA4) hydrolase activity. To characterize this activity further, airway epithelial cells, cultured to confluence, were disrupted by sonication and were fractionated at 15,000 and 100,000 x g. Enzymatic activity was assessed by incubating fractions with 15 microM LTA4 at 37 degrees C for 15 min. LTA4 hydrolase activity was present in the 15,000 x g and the 100,000 x g supernatants and was inactivated by heating at 56 degrees C or by pronase, as is the case for neutrophil LTA4 hydrolase. However, the epithelial cell enzyme had a slower 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 consistent with an enzyme that has multiple active sites. Immunoblotting, performed with anti-neutrophil LTA4 hydrolase antibody, recognized 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 chromatography of epithelial cell samples revealed that LTA4 hydrolase and aminopeptidase activity did not co-elute, whereas one of three peaks of aminopeptidase activity did co-elute in chromatograms of neutrophil samples. Immunoblots of proteolytic digests of partially purified M(r) 69,000 protein from epithelial cells and neutrophils revealed different immunoreactive bands. The digest of the M(r) 110,000-115,000 protein revealed no immunoreactive bands. Repeat kinetic analysis on 179-fold purified epithelial LTA4 hydrolase again revealed that it lacked significant aminopeptidase activity and retained its unique kinetic properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Bigby
T D
TD
Veterans Affairs Medical Center, San Diego, California 92161.
Lee
D M
DM
Minami
M
M
Ohishi
N
N
Shimizu
T
T
Baker
J R
JR
D018826:CD13 Antigens / Q000378:metabolism; D002458:Cell Fractionation; D002478:Cells, Cultured; D004847:Epithelial Cells; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D017572:Leukotriene A4 / Q000378:metabolism; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology; D012137:Respiratory System / Q000201:enzymology*
D017572:Leukotriene A4; D004851:Epoxide Hydrolases; D018826:CD13 Antigens; C043134:leukotriene A4 hydrolase