Purification and characterization of Ca(2+)-dependent phospholipases A2 from rat kidney.
Three phospholipase A2 (PLA2) activities were identified in rat kidney. In the particulate fraction a PLA2 activity was present which was cross-reactive with polyclonal antibodies against the 14-kDa group II PLA2. This PLA2 was partially solubilized and purified to near homogeneity. The amino acid sequence at the N-terminus of the purified enzyme was identical to that of the 14-kDa rat group II PLA2 from rat liver mitochondria, platelet, and spleen. The cytosolic fraction of rat kidney contained at least two PLA2 activities which could be separated on a Mono Q column. Upon gel filtration the activity that eluted from the anion-exchange column in the salt gradient behaved as a high molecular mass PLA2, exhibited a preference for arachidonic acid at the sn-2 position of glycerophospholipids, and was already optimally active at submillimolar Ca2+ concentrations. The cytosolic PLA2 activity that did not bind to the anion-exchange column was purified by gel filtration, immunoaffinity chromatography using immobilized polyclonal antibodies to group I PLA2, and C18 reversed-phase chromatography. Immunological properties and N-terminal sequence analysis identified this enzyme as rat group I PLA2. Rat glomerular mesangial cells contained only group II and high molecular mass PLA2 enzymes.
Aarsman
A J
AJ
Centre for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
Schalkwijk
C G
CG
Neys
F W
FW
Iijima
N
N
Wherrett
J R
JR
van den Bosch
H
H
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D013379:Substrate Specificity
D010446:Peptide Fragments; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium