1. Subcellular fractions of bovine brain degrade phosphatidylcholine by sequential deacylation of the sn-1 and sn-2 positions
- Author
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David W. Wu, Matthew J. Pete, and John H. Exton
- Subjects
Coenzyme A ,Acylation ,Biophysics ,Biochemistry ,Phospholipases A ,chemistry.chemical_compound ,Endocrinology ,Phospholipase A1 ,Phosphatidylcholine ,Animals ,Phospholipase A ,Chromatography ,Chemistry ,Hydrolysis ,Brain ,Lysophosphatidylcholines ,Phospholipid Ethers ,Metabolism ,Phospholipases A1 ,Lysophospholipase ,Phosphatidylcholines ,lipids (amino acids, peptides, and proteins) ,Arachidonic acid ,Cattle ,PMSF ,Subcellular Fractions - Abstract
Phosphatidylcholine (PC) metabolism was investigated using cytosol (fraction I) and particulate fractions of bovine brain that were enriched with microsomes (fraction II), plasma membranes (fraction III) or mitochondria (fraction IV). Fractions I-III incubated with 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine yielded [14C]arachidonic acid at near equal rates, whereas only fraction I accumulated significant amounts of 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine. Much slower rates of arachidonic acid release were observed using an ether PC (1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine). Moreover, arachidonic acid yield from the diacyl, but not ether PC was slowed by pretreating fractions I-III, but not IV, with phenylmethylsulfonyl fluoride (PMSF). Coincident with this decreased arachidonic acid, 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine was increased, indicating high PLA1 activity. Taken together these data suggest that arachidonic release was largely dependent on initial deacylation of position sn-1. Incubating each untreated fraction with 2-[3-H]arachidonoyl-sn-glycero-3-phosphocholine yielded [3H]arachidonic acid (lysophospholipase A2 activity) at rate that was substantially greater than that using the comparable PMSF-treated fraction. Thus, the large effect of PMSF on arachidonic acid release can be accounted for if much of the fatty acid formation arose from the sequential sn-1 and sn-2 deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2. When PMSF-treated fractions were incubated with 2-[3H]arachidonoyl-sn-glycero-3-phosphocholine, [3H]PC accumulated at low rates that were enhanced by adding coenzyme A or stearoyl-coenzyme A. Thus, the lysophospholipid was also reacylated to form PC, but this reaction was negligible in the absence of PMSF and added cofactors. In summary, we conclude that, in brain subcellular fractions, deacylation of the sn-1 position of diacyl-PC proceeded more rapidly than sn-2 hydrolysis. There was substantial further metabolism of 2-acyl lysophospholipids due to the combined activities of a PMSF-sensitive and -insensitive lysophospholipase. Finally, the sequential deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2 probably accounted for the major portion of arachidonic acid produced.
- Published
- 1996