Human keratinocytes possess an sn-2 acylhydrolase that is biochemically similar to the U937-derived 85-kDa phospholipase A2.
The phospholipase A2 (PLA2) activities that are localized in the keratinocyte cytosolic and microsomal fractions were biochemically and pharmacologically characterized. The cytosol and to a lesser extent the microsome were sensitive to heat treatment and stable in the presence of sulfhydryl reducing agents. Both fractions were almost totally inactivated by reduction of pH to 2. The cytosolic activity demonstrated a sevenfold preference for arachidonic acid over oleic acid in the sn-2 position of substrate phospholipid and the microsome exhibited a fourfold preference. Neither the cytosol nor the microsome was inactivated by a neutralizing mouse monoclonal antibody 3F10 generated against recombinant human (rh) type II 14-kDa PLA2. Western immunoblot analysis of both fractions identified a high-molecular-mass protein in keratinocyte cytosol but not the microsome that migrated with rh 85-kDa PLA2. Neither the cytosol nor the microsome possessed immunoreactive bands that migrated with rh type II 14-kDa PLA2 when probed with monoclonal antibody 3F10. Further analysis of the cytosolic activity showed that it was activated by submicromolar concentrations of Ca2+, reduced by arachidonyl trifloromethylketone, a selective 85-kDa PLA2 inhibitor, but was unaffected by C-7 phosphonate phospholipid, a selective 14-kDa PLA2 transition state inhibitor. Taken together, the data supports the existence of a PLA2 activity in the cytosol that displays characteristics that are indistinguishable from those exhibited by the 85-kDa PLA2. Alternatively, both the cytosol and microsome were devoid of type II 14-kDa-like PLA2 activity. The failure of 12-epi scalaradial, a 14-kDa PLA2 inhibitor, to modify A23187-stimulated keratinocyte prostaglandin E2 release, was consistent with the biochemistry and suggests that the 85-kDa PLA2 may play an important role in keratinocyte prostaglandin E2 formation.
McCord
M
M
Department of Inflammation and Respiratory Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Chabot-Fletcher
M
M
Breton
J
J
Marshall
L A
LA
D000911:Antibodies, Monoclonal; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D015232:Dinoprostone / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010741:Phospholipases A / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D054467:Phospholipases A2; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000001:Calcimycin; D010741:Phospholipases A; D054467:Phospholipases A2; D015232:Dinoprostone