ISOLATION AND CHARACTERIZATION OF A CYTOSOLIC PHOSPHOLIPASE A(2) FROMBOVINE ADRENAL-MEDULLA

We have recently demonstrated that bovine adrenal medulla contains a s oluble phospholipase A(2) (PLA(2)), which is localized in the cytosol. In the present study, this PLA(2) was purified 1,097-fold using seque ntial concanavalin A, hydrophobic interaction, anion exchange, gel fil tration, and an additional anion exchange chromatography. The enzyme i s activated over the range of 20-1,000 mu M Ca2+ and has a pH optimum near 8.0. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis , the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA(2) is no t inhibited by NaCl, and the enzymatic activity is stimulated at low c oncentrations of Triton X-100 (0.01%) and deoxycholate (1 mM) but inhi bited at higher concentrations (0.1% and 3 mM, respectively) of these detergents. Furthermore, heat treatment (57 degrees C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the ac tivity. p-Bromophenacylbromide, a frequently used irreversible inhibit or of type II PLA(2), has little effect until 100 mu M, and 2-10 mM di thiothreitol totally inactivated the enzyme. The purified PLA(2) displ ays a preference for phosphatidyl-choline as a substrate but hydrolyze s phospholipid substrates with arachidonic acid or linoleic acid ester ified at the sn-2 position to the same extent. It is concluded that th e chromaffin cell cytosolic PLA(2), which was isolated and characteriz ed in this study, represents a type of PLA(2) that has not been former ly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA(2) will help to reveal whether the enzyme is impor tant for exocytosis.