TY - JOUR
T1 - Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2
AU - Wolf, Matthew J.
AU - Gross, Richard W.
PY - 1996/12/14
Y1 - 1996/12/14
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0029804203&partnerID=8YFLogxK
U2 - 10.1074/jbc.271.48.30879
DO - 10.1074/jbc.271.48.30879
M3 - Article
C2 - 8940072
AN - SCOPUS:0029804203
VL - 271
SP - 30879
EP - 30885
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 48
ER -