Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine.
We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.
Hirashima
Y
Y
Department of Physiological Chemistry, Ohio State University, Columbus 43210.
Mills
J S
JS
Yates
A J
AJ
Horrocks
L A
LA
D000310:Adrenal Gland Neoplasms / Q000201:enzymology; D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure; D002417:Cattle; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000201:enzymology; D005910:Glioma / Q000201:enzymology; D006851:Hydrochloric Acid; D009380:Neoplasms, Nerve Tissue / Q000201:enzymology*; D009447:Neuroblastoma / Q000201:enzymology; D010673:Pheochromocytoma / Q000201:enzymology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010955:Plasmalogens / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D010955:Plasmalogens; C020791:phosphatidal ethanolamines; D010741:Phospholipases A; D054467:Phospholipases A2; D006851:Hydrochloric Acid