HUMAN KERATINOCYTES POSSESS AN SN-2 ACYLHYDROLASE THAT IS BIOCHEMICALLY SIMILAR TO THE U937-DERIVED 85-KDA PHOSPHOLIPASE A(2)

The phospholipase A(2) (PLA(2)) 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 PLA(2). 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 PLA(2). Neither the sytosol nor the microsome possessed immunoreative bands that migrated with rh type II 14-kDa PLA(2) 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 PLA(2) inhibitor, but was unaffected by C-7 phosphonate phospholipid, a selective 14-kDa PLA(2) transition state inhibitor. Taken together, the data supports the existence of a PLA(2) activity in the cytosol that displays characteristics that are indistinguishable from those exhibited by the 85-kDa PLA(2). Alternatively, both the cytosol and microsome were devoid of type II 14-kDa- like PLA(2) activity. The failure of 12-epi scalaradial, a 14-kDa PLA(2) inhibitor, to modify A23187-stimulated keratinocyte prostaglandin E(2) release, was consistent with the biochemistry and suggests that the 85-kDa PLA(2) may play an important role in keratinocyte prostaglandin E(2) formation.