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Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils
Author(s) -
Smith Duane M.,
Waite Moseley
Publication year - 1992
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.52.6.670
Subject(s) - phosphatidylinositol , biology , phospholipase c , peripheral blood , phospholipase , phospholipase a2 , hydrolysis , immunology , human blood , phos , phospholipase d , biochemistry , microbiology and biotechnology , enzyme , signal transduction , physiology
We describe here and partially characterize a Ca 2+ ‐independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo‐[ 3 H]inositol to form [ 3 H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [ 3 H]lysophos‐ phatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [ 3 H]arachi‐ donic acid from exogenous [l‐ 14 C]stearoyl‐2‐[ 3 H]arachi‐ donyl‐phosphatidylinositol. This phospholipase A2 is Ca 2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol‐hydrolyzing phospholipase C activity was characterized by the production of [ 3 H]‐/[ 14 C]‐diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca 2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/ cyclooxygenase inhibitors eicosatetrayenoic acid and nor‐ dihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol‐hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, mono‐ glyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane‐enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N‐formyl‐ methionyl‐leucyl‐phenylalanine, and then subfraction‐ ated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane‐ enriched fractions. These data suggest that this Ca 2+ ‐ independent, phosphatidylinositol‐hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.