Isolation and characterization of phospholipase A2 from rat lung with affinity chromatography and two-dimensional gel electrophoresis.
A phospholipase A2 (PLA2, EC 3.1.1.4) from rat lung has been isolated and characterized. PLA2 was purified with ion-exchange and affinity chromatography and the purified enzyme was characterized with regard to pH optimum and calcium dependence. The isolated enzyme was also analyzed with two-dimensional gel electrophoresis and identified by Western immunoblots. The enzyme activity was found to be highest at pH 9.5-10.0, with a requirement for calcium, and the molecular mass was estimated to be 12 kDa by means of SDS-polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis analysis revealed two isoforms of PLA2 with isoelectric points of 7.8 and 9.5. On DEAE-Sepharose, PLA2 eluted as two peaks, one in the flow-through fraction and the other with increased salt concentration. Both peaks contained the same two PLA2 isoforms, as judged by two-dimensional gel electrophoresis. These results demonstrate the presence in rat lung of two isoforms of a calcium-dependent 12 kDa PLA2 with alkaline pH optimum. Using two-dimensional gel electrophoresis, this enzyme can be identified also in rat bronchoalveolar lavage fluid.
Lindahl
M
M
Department of Occupational Medicine, Linköping University, Sweden.
von Schenck
H
H
Tagesson
C
C
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D011919:Rats, Inbred Strains
D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2