Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2.
Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.
Aarsman
A J
AJ
Centre for Biomembranes and Lipid Enzymology, State University of Utrecht, The Netherlands.
de Jong
J G
JG
Arnoldussen
E
E
Neys
F W
FW
van Wassenaar
P D
PD
Van den Bosch
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification*; D054467:Phospholipases A2; D051381:Rats; D013045:Species Specificity; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2