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J. Biol. Chem., Vol. 263, Issue 7, 3079-3085, Mar, 1988
RJ Ulevitch, Y Watanabe, M Sano, MD Lister, RA Deems and EA Dennis
The release of free arachidonic acid from membrane phospholipids is
believed to be the rate-controlling step in the production of the
prostaglandins, leukotrienes, and related metabolites in inflammatory cells
such as the macrophage. We have previously identified several different
phospholipases in the macrophage-like cell line P388D1 potentially capable
of controlling arachidonic acid release. Among them, a membrane-bound,
alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular
interest because of the likelihood that the regulatory enzyme has these
properties. This phospholipase A2 has now been solubilized from the
membrane fraction with octyl glucoside and partially purified. The first
two steps in this purification are butanol extractions that yield a
lyophilized, stable preparation of phospholipase A2 lacking other
phospholipase activities. This phospholipase A2 shows considerably more
activity when assayed in the presence of glycerol, regardless of whether
the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated
vesicles or mixed micelles with the nonionic surfactant Triton X-100.
Glycerol (70%) increases both the Vmax and the Km with both substrate
forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of
about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an
apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater
for vesicles than for mixed micelles. The lyophilized preparation of the
enzyme is routinely purified about 60-fold and is suitable for evaluating
phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps
in the purification are acetonitrile extraction followed by high
performance liquid chromatography on an Aquapore BU-300 column and a
Superose 12 column. This yields a 2500- fold purification of the
membrane-bound phospholipase A2 with a 25% recovery and a specific activity
of about 800 nmol min-1 mg-1 toward 100 microM
dipalmitoylphosphatidylcholine in mixed micelles. When this material was
subjected to analysis on a Superose 12 sizing column, the molecular mass of
the active fraction was approximately 18,000 daltons.
Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
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