Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2

Matthew J. Wolf, Richard W. Gross

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Abstract

A CHO cell-derived 80-kDa recombinant polypeptide (GenBank number I15470) putatively encoding a calcium-independent phospholipase A2 was expressed in S. frugiperda cells resulting in over a 15-fold increase in a calcium-independent phospholipase A1/A2 activity which was entirely inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran- 2-one. The recombinant polypeptide was purified from cytosol by sequential tandem affinity chromatographies employing ATP-agarose and calmodulin- Sepharose stationary phases. This strategy resulted in the rapid purification (36 h) of recombinant phospholipase A2 activity in 56% overall yield to a single intense 80-kDa protein band on SDS-polyacrylamide gel electrophoresis after silver staining. The purified protein possessed phospholipase A1, phospholipase A2, and lysophospholipase activities. Microbore anion exchange chromatography demonstrated that the 80-kDa protein band was comprised of multiple distinct isoforms including an anionic isoform which possessed over a 5-fold higher specific activity (5 μmol/mg · min) than earlier eluting isoforms. Collectively, these results unambiguously demonstrate that: 1) the 80-kDa polypeptide catalyzes phospholipase A1/A2 and lysophospholipase activities with distinct kinetic parameters; 2) calmodulin and ATP both interact with the catalytic polypeptide independent of regulatory proteins; and 3) distinct isoforms of this polypeptide exist which possess markedly different specific activities.

Original languageEnglish
Pages (from-to)30879-30885
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number48
DOIs
StatePublished - Dec 14 1996

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