E. Lamura et al., COMPARTMENTALIZATION AND CHARACTERISTICS OF A CA2-DEPENDENT PHOSPHOLIPASE A(2) IN HUMAN COLON MUCOSA(), Biochemical pharmacology, 53(9), 1997, pp. 1323-1332
The biochemical properties of the phospholipase A(2) (PLA(2)) found in
the 100,000 x g centrifugate cytosol or particulate fractions of huma
n colonic mucosa have been investigated using both deoxycholate-solubi
lized and Escherichia coli (E. coli) phospholipids as substrates. PLA(
2) activity was present in both subcellular fractions and the profiles
of biochemical activites were similar. Activity in the particulate fr
action was approximately twofold greater than the cytosol fraction whe
n expressed on the basis of protein concentration. The PLA, is Ca2+ de
pendent and using EGTA regulated buffers cytosolic or particulate frac
tion activity was similar at both 10 mu m or 10 mm Ca2+ concentrations
. Using deoxycholate phospholipid micelles as substrate a small but st
atistically significant twofold preference for glycero-phosphatidylcho
line bearing sn-2 arachidonate compared with sn-2-oleate was seen, but
this preference was not noted using arachidonate or oleate labelled E
. coli membranes. Dithiothreitol (10 mM) reduced colon mucosal cytosol
PLA(2) activity significantly by 63.5 +/- 1.90% in cytosol and by 30.
54 +/- 1.27% in microsomes using micelles as substrate or by 84.3 +/-
2.30% in cytosol and by 69.33 +/- 11.30% in microsomes using oleate-la
belled E. coli as substrates. Warming at 57 degrees C reduced activity
significantly by 35.0 +/- 5.80% in microsomes and by 40.0 +/- 1.08% i
n cytosol. Acid treatment increased PLA(2) activity to 148 +/- 16.3% i
n microsomes and 145 +/- 18.6% in cytosol. When mucosal preparations w
ere subjected to heparin Sepharose chromatography, it bound tightly an
d eluted in the same position on a salt gradient as authentic human gr
oup II PLA(2). Further purification by gel-permeation chromatography g
ave activity in the 14 kDa region of the elution profile. These featur
es have many of the characteristics expected of a 14 kDa isoform of PL
A(2) but exhibit activity at concentrations of Ca2+ that are relevant
in the intracellular environment and may participate in cellular lipid
metabolism. (C) 1997 Elsevier Science Inc.