Isolation and characterization of a cytosolic phospholipase A2 from bovine adrenal medulla.
We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A2 (PLA2), which is localized in the cytosol. In the present study, this PLA2 was purified 1,097-fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20-1,000 microM 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 PLA2 is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X-100 (0.01%) and deoxycholate (1 mM) but inhibited 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 activity. p-Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA2, has little effect until 100 microM, and 2-10 mM dithiothreitol totally inactivated the enzyme. The purified PLA2 displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn-2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA2, which was isolated and characterized in this study, represents a type of PLA2 that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA2 will help to reveal whether the enzyme is important for exocytosis.
Petit
K
K
Department of Medicine, University of Antwerp, Wilrijk, Belgium.
De Block
J
J
De Potter
W
W
D000098:Acetophenones / Q000494:pharmacology; D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002838:Chromaffin System / Q000166:cytology / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology*; D003840:Deoxycholic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D017830:Octoxynol / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
D000098:Acetophenones; D010743:Phospholipids; D003840:Deoxycholic Acid; D012965:Sodium Chloride; D017830:Octoxynol; D010741:Phospholipases A; D054467:Phospholipases A2; D005990:Glycerol; C006821:4-bromophenacyl bromide; D002118:Calcium