COMPARTMENTALIZATION AND CHARACTERISTICS OF A CA2-DEPENDENT PHOSPHOLIPASE A(2) IN HUMAN COLON MUCOSA()

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.