Compartmentalisation and characteristics of a Ca2+-dependent phospholipase A2 in human colon mucosa.
The biochemical properties of the phospholipase A2 (PLA2) found in the 100,000 x 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. PLA2 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 PLA2 is Ca2+ dependent and using EGTA-regulated buffers cytosolic or particulate fraction activity was similar at both 10 microm or 10 mm Ca2+ 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 PLA2 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-labelled E. coli as substrates. Warming at 57 degrees C reduced activity significantly by 35.0 +/- 5.80% in microsomes and by 40.0 +/- 7.08% in cytosol. Acid treatment increased PLA2 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 PLA2. 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 PLA2 but exhibit activity at concentrations of Ca2+ that are relevant in the intracellular environment and may participate in cellular lipid metabolism.
Lamura
E
E
Clinical Pharmacology Group, University of Southampton, UK.
Hillier
K
K
Kinkaid
A
A
Wilton
D
D
D000328:Adult; D000368:Aged; D002118:Calcium / Q000494:pharmacology*; D003106:Colon / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008875:Middle Aged; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium; D004229:Dithiothreitol