The detection of multimeric forms of phospholipase A2 upon sodium dodecylsulfate-polyacrylamide electrophoresis.
Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were found to be cross-reactive with the phospholipase A2 present in caseinate-induced rat peritoneal exudate, both in dot-blot and in monoclonal antibody-Sepharose binding experiments. Immunoaffinity purification of the exudate enzyme yielded enzyme preparations with an estimated enrichment of about 54000-fold in a single chromatographic step. Sodium dodecylsulfate-polyacrylamide gelectrophoresis showed, apart from the presence of a 14 KDa phospholipase A2 band, several other bands at higher molecular weights. After Western blotting and immunostaining only a 14 kDa phospholipase A2 was detected, but upon storage of the sample in dodecylsulfate and dithiotreitol associated forms of this phospholipase A2 appeared. Similar tendencies to form multimers were observed starting from immunopurified and homogeneous phospholipase A2 preparations obtained from rat platelets and from rat liver mitochondria. This phenomenon is likely to be of general importance for the interpretation of results obtained in Western blots using antiphospholipase A2 antibodies.
Schalkwijk
C G
CG
Centre for Biomembranes and Lipid Enzymology, University of Utrecht, The Netherlands.
Märki
F
F
Wiesenberg
I
I
van den Bosch
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001202:Ascitic Fluid / Q000201:enzymology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D010741:Phospholipases A / Q000032:analysis* / Q000276:immunology; D054467:Phospholipases A2; D051381:Rats; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D012967:Sodium Dodecyl Sulfate; D010741:Phospholipases A; D054467:Phospholipases A2