TY - JOUR
T1 - Purification and characterization of canine myocardial cytosolic phospholipase A2
T2 - A calcium-independent phospholipase with absolute sn-2 regiospecipicity for diradyl glycerophospholipids
AU - Hazen, S. L.
AU - Stuppy, R. J.
AU - Gross, R. W.
PY - 1990/6/25
Y1 - 1990/6/25
N2 - Recently, we identified a novel calcium-independent, plasmalogen-selective phospholipase A2 activity in canine myocardial cytosol which represents the major measurable phospholipase A2 activity in myocardial homogenates (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303). We now report the 154,000-fold purification of this phospholipase A2 to homogeneity through utilization of sequential anion exchange, chromatofocusing, affinity, Mono Q, and hydroxylapatite chromatographies. The purified enzyme had a molecular mass of 40 kDa, possessed a specific activity of 227 μmol/mg min, had a pH optimum of 6.4, and catalyzed the regiospecific cleavage of the sn-2 fatty acid from diradyl glycerophospholipids. The purified polypeptide was remarkable for its ability to selectively hydrolyze plasmenylcholine in homogeneous vesicles (subclass rank order: plasmenylcholine > alkyl-ether choline glycerophospholipid > phosphatidylcholine) as well as in mixed bilayers comprised of equimolar plasmenylcholine/phosphatidylcholine. Purified myocardial phospholipase A2 also possessed selectivity for hydrolysis of phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic or palmitic acid. Taken together, these results constitute the first purification of a calcium-independent phospholipase with absolute regiospecificity for cleavage of the sn-2 acyl linkage in diradyl glycerophospholipids and demonstrate that myocardial phospholipase A2 has kinetic characteristics which are anticipated to result in the selective hydrolysis of sarcolemmal phospholipids during myocardial ischemia.
AB - Recently, we identified a novel calcium-independent, plasmalogen-selective phospholipase A2 activity in canine myocardial cytosol which represents the major measurable phospholipase A2 activity in myocardial homogenates (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303). We now report the 154,000-fold purification of this phospholipase A2 to homogeneity through utilization of sequential anion exchange, chromatofocusing, affinity, Mono Q, and hydroxylapatite chromatographies. The purified enzyme had a molecular mass of 40 kDa, possessed a specific activity of 227 μmol/mg min, had a pH optimum of 6.4, and catalyzed the regiospecific cleavage of the sn-2 fatty acid from diradyl glycerophospholipids. The purified polypeptide was remarkable for its ability to selectively hydrolyze plasmenylcholine in homogeneous vesicles (subclass rank order: plasmenylcholine > alkyl-ether choline glycerophospholipid > phosphatidylcholine) as well as in mixed bilayers comprised of equimolar plasmenylcholine/phosphatidylcholine. Purified myocardial phospholipase A2 also possessed selectivity for hydrolysis of phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic or palmitic acid. Taken together, these results constitute the first purification of a calcium-independent phospholipase with absolute regiospecificity for cleavage of the sn-2 acyl linkage in diradyl glycerophospholipids and demonstrate that myocardial phospholipase A2 has kinetic characteristics which are anticipated to result in the selective hydrolysis of sarcolemmal phospholipids during myocardial ischemia.
UR - http://www.scopus.com/inward/record.url?scp=0025346796&partnerID=8YFLogxK
M3 - Article
C2 - 2355013
AN - SCOPUS:0025346796
SN - 0021-9258
VL - 265
SP - 10622
EP - 10630
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 18
ER -