2024-03-28T18:30:18Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1773552020-05-20T12:17:49Zcom_10261_86com_10261_1col_10261_339
DIGITAL.CSIC
author
Balsinde, Jesús
author
Díez, Emilio
author
Mollinedo, Faustino
funder
Dirección General de Investigación Científica y Técnica, DGICT (España)
orcid
Balsinde, Jesús [0000-0002-4157-6714]
orcid
Mollinedo, Faustino [0000-0002-4939-2434]
2019-03-06T11:07:57Z
2019-03-06T11:07:57Z
1991-08-25
The Journal of Biological Chemistry 266:15638-15643 (1991)
0021-9258
http://hdl.handle.net/10261/177355
1083-351X
http://dx.doi.org/10.13039/501100008737
We have studied the capacity of human neutrophils to release arachidonic acid from diacylglycerol, employing 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol and 1-[1-14C]stearoyl-2-arachidonoyl-sn-glycerol as exogenous substrates. We have found that arachidonic acid is removed from diacylglycerol by the sequential action of two enzymes. First, the sn-1 position is split by 1-diacylglycerol lipase activity, and then, arachidonic acid is released from the resulting 2-monoacylglycerol by a 2-monoacylglycerol lipase. The specific activity of the 2-monoacylglycerol lipase, using 2-[1-14C]arachidonoyl-sn-glycerol as exogenous substrate, was at least 9-fold higher than that of 1-diacylglycerol lipase, indicating that the action of the 1-diacylglycerol lipase is the rate-limiting step in arachidonic acid release from diacylglycerol. Postnuclear supernatants from A23187-treated cells showed a 2.5-fold increase in both lipase activities. The arachidonic acid-releasing diacylglycerol lipase system showed an optimum pH of 4.5 and was not inhibited by EGTA or stimulated by Ca2+, Mg2+, Mn2+, Zn2+, or Co2+. However, arachidonic acid release was inhibited by Hg2+, suggesting the involvement of sulfhydryl groups in catalytic activity. The subcellular distribution of both 1-diacylglycerol lipase and 2-monoacylglycerol lipase activities was examined in resting and A23187-treated human neutrophils by fractionation of postnuclear supernatants on continuous sucrose gradients. Both lipases were localized mainly in the membrane of gelatinase-containing granules, which were resolved from cytosol, plasma membrane, phosphasomes, and specific and azurophilic granules. When neutrophils were stimulated by the calcium ionophore A23187, a drastic shift of the 1-diacylglycerol lipase and 2-monoacylglycerol lipase toward the plasma membrane was detected. This shift was due to fusion of gelatinase-containing granules with the plasma membrane upon neutrophil stimulation. As a result of the membrane fusion process, the capacity to release arachidonic acid from diacylglycerol was increased. This translocation from the membrane of gelatinase-containing granules to the plasma membrane may play an important role in regulating the diacylglycerol level in stimulated human neutrophils.
eng
openAccess
Arachidonic acid release from diacylglycerol in human neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an intracellular pool to plasma membrane upon cell activation
artículo
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URL
https://digital.csic.es/bitstream/10261/177355/1/J.%20Biol.%20Chem.-1991-Balsinde-15638-43.pdf
File
MD5
37dedcb12ce44ca3a00eefede554321a
837714
application/pdf
J. Biol. Chem.-1991-Balsinde-15638-43.pdf