Characterization of a triacylglycerol lipase that liberates arachidonic acid from bovine chromaffin cells during secretion.
Primary cultures of chromaffin cells from bovine adrenal medullae were used as a model to study lipolytic events during stimulus-secretion coupling. It has been shown that chromaffin cells liberate arachidonic acid in addition to their main secretion product, the catecholamines. To understand more about the mechanism of arachidonic acid liberation, chromaffin cells were labeled with radioactive arachidonic acid, stimulated, and then analyzed for changes in lipid composition. After stimulation with 10(-4) M acetylcholine, the radioactivity of triacylglycerols decreased to the same extent that the free arachidonic acid level rose. This finding suggests that in bovine chromaffin cells a stimulation-dependent triacylglycerol lipase (triacylglycerol hydrolase; EC 3.1.1.3) is involved in arachidonic acid liberation. Further work was performed on detection, characterization, and isolation of this enzyme. Triacylglycerol lipase activity was found in whole cell homogenates and in plasma membrane fractions isolated from adrenal medullary tissue. The plasma membrane lipase showed a pH optimum of 4.3. The apparent Michaelis constant was determined as 3.3 x 10(-4) mol/L. Ca2+ did not influence the enzymatic activity. To differentiate the plasma membrane triacylglycerol lipase from the previously described plasma membrane diacylglycerol lipase of chromaffin cells, the influence of RG 80267, a specific diacylglycerol lipase inhibitor, was examined. RG 80267 (50 microM) inhibited the triacylglycerol lipase by only 24%, although diacylglycerol lipase was totally inhibited with only 20 microM RG 80267. The pH optimum of homogenate lipase was broad, lying between 4 and 7. Starting from the soluble fraction of whole cell homogenates, the triacylglycerol lipase was partially purified by ultracentrifugation and size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Galatioto
L E
LE
Institute of Biochemistry, University of Berne, Switzerland.
Zahler
P
P
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D008049:Lipase / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D016718:Arachidonic Acid; D008049:Lipase