Purification and characterization of a phospholipase A2 associated with rabbit lung microsomes: some evidence for its mitochondrial origin.
Phospholipase A2 (EC 3.1.1.4) activity appeared to be unevenly distributed among the subcellular fractions of rabbit lung homogenates. The mitochondrial/lysosomal fraction, which possessed the highest specific activity, was the second most abundant source of enzyme, following the 1000 x g pellet. Crude microsomes, which were the poorest source of enzyme, had a specific activity intermediate between that of crude mitochondria and of cytosol. Despite these observations, in view of the putative role of microsomal phospholipase A2 in remodelling phosphatidylcholines for pulmonary surfactant biosynthesis, the purification of phospholipase A2 from microsomal membranes was investigated. The activity was solubilized from rabbit lung microsomes with 1 M KCl and resolved into two distinct peaks by ion-exchange chromatography. The larger peak (95% of the recovered activity) was subjected to a combination of hydroxyapatite and gel-filtration chromatography, resulting in a purification factor in excess of 70,000 relative to the microsomal membranes. There was no indication for the removal of endogenous inhibitor(s) during the purification. Application of the same purification protocol to a 1 M KCl extract of lung mitochondria resulted in phospholipase A2 profiles in each of the four columns employed that had exactly the same elution characteristics as those generated by the microsomal extracts. The purified enzyme is specific for the sn-2 ester bond of phosphatidylcholine, requires Ca2+ for activity and has an alkaline pH optimum. It is heat-labile and susceptible to treatment by p-bromophenacyl bromide and by 2-mercaptoethanol but remains unaffected by NaF, diisopropylfluorophosphate and thiol reagents.
Filgueiras
O M
OM
MRC Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Canada.
Possmayer
F
F
D000818:Animals; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000187:drug effects / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification*; D054467:Phospholipases A2; D011817:Rabbits; D013379:Substrate Specificity
D006882:Hydroxyapatites; D010713:Phosphatidylcholines; D007531:Isoflurophate; D008623:Mercaptoethanol; D010741:Phospholipases A; D054467:Phospholipases A2