Td. Bigby et al., CHARACTERIZATION OF HUMAN AIRWAY EPITHELIAL-CELL LEUKOTRIENE A(4) HYDROLASE, American journal of respiratory cell and molecular biology, 11(5), 1994, pp. 615-624
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.