Aj. Aarsman et al., PURIFICATION AND CHARACTERIZATION OF CA2-DEPENDENT PHOSPHOLIPASES A(2) FROM RAT-KIDNEY(), Archives of biochemistry and biophysics, 331(1), 1996, pp. 95-103
Three phospholipase A(2) (PLA(2)) activities were identified in rat ki
dney, In the particulate fraction a PLA(2) activity was present which
was cross-reactive with polyclonal antibodies against the 14-kDa group
II PLA(2). This PLA(2) was partially solubilized and purified to near
homogeneity. The amino acid sequence at the N-terminus of the purifie
d enzyme was identical to that of the 14-kDa rat group II PLA(2) from
rat liver mitochondria, platelet, and spleen. The cytosolic fraction o
f rat kidney contained at least two PLA(2) activities which could be s
eparated on a Mono Q column. Upon gel filtration the activity that elu
ted from the anion-exchange column in the salt gradient behaved as a h
igh molecular mass PLA(2), exhibited a preference for arachidonic acid
at the sn-2 position of glycerophospholipids, and was already optimal
ly active at submillimolar Ca2+ concentrations. The cytosolic PLA(2) a
ctivity that did not bind to the anion-exchange column was purified by
gel filtration, immunoaffinity chromatography using immobilized polyc
lonal antibodies to group I PLA(2), and C18 reversed-phase chromatogra
phy. Immunological properties and N-terminal sequence analysis identif
ied this enzyme as rat group I PLA(2). Rat glomerular mesangial cells
contained only group II and high molecular mass PLA(2) enzymes. (C) 19
96 Academic Press, Inc.