Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2.
A CHO cell-derived 80-kDa recombinant polypeptide (GenBank number I15470I15470) 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 micromol/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.
Wolf
M J
MJ
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gross
R W
RW
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054466:Phospholipases A1; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D007527:Isoenzymes; D011994:Recombinant Proteins; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2; D002118:Calcium