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Cobra venom phospholipase A2 immobilized to porocus glass beads.
Authors:M Adamich  H F Voss  E A Dennis
Institution:Department of Chemistry, University of California at San Diego, La Jolla, California 92093 USA
Abstract:Phospholipase A2 (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A2 toward phosphatidylcholine is about 160 μmol min?1 ml?1 of glass beads, and the specific activity is about 13 μmol min?1 mg?1 of protein in this assay system. The pH rate profile and apparent pKa in 10 mm Ca2+ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A2 from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.
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