Compartmentalisation and characteristics of a CA 2+-dependent phospholipase A 2 in human colon mucosa

Abstract

The biochemical properties of the phospholipase A 2 (PLA 2) found in the 100,000 × g centrifugate cytosol or particulate fractions of human colonic mucosa have been investigated using both deoxycholate-solubilized 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 fraction was approximately twofold greater than the cytosol fraction when expressed on the basis of protein concentration. The PLA 2 is Ca 2+ dependent and using EGTA-regulated buffers cytosolic or particulate fraction activity was similar at both 10 μm or 10 mm Ca 2+ concentrations. Using deoxycholate-phospholipid micelles as substrate a small but statistically significant twofold preference for glycero-phosphatidylcholine 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 843 ± 2.30% in cytosol and by 69.33 ± 11.30% in microsomes using oleate-labelled E. coli as substrates. Warming at 57 °C reduced activity significantly by 35.0 ± 5.80% in microsomes and by 40.0 ± 7.08% in cytosol. Acid treatment increased PLA 2 activity to 148 ± 16.3% in microsomes and 145 ± 18.6% in cytosol. When mucosal preparations were subjected to heparin-Sepharose chromatography, it bound tightly and eluted in the same position on a salt gradient as authentic human group II PLA 2. Further purification by gel-permeation chromatography gave activity in the 14 kDa region of the elution profile. These features have many of the characteristics expected of a 14 kDa isoform of PLA 2 but exhibit activity at concentrations of Ca 2+ that are relevant in the intracellular environment and may participate in cellular lipid metabolism.