Molecular Species of Ethanolamine Phosphatides of Dog and Pig Kidney

In brain, phosphatidylethanolamine can be synthesized from free ethanolamine either by a pathway involving the formation of CDP-ethanolamine and its transfer to diglyceride, or by base-exchange of ethanolamine with existing phospholipids. Although de novo synthesis from serine has also been demonstrated, the metabolic pathway involved is not known. The enzyme phosphatidylserine decarboxylase appears to be involved in the synthesis of much of the phosphatidylethanolamine in liver, but the significance of this route in brain has been challenged. Our in vitro studies demonstrate the existence of phosphatidylserine decarboxylase activity in rat brain and characterize some of its properties. This enzyme is localized in the mitochondrial fraction, whereas the enzymes involved in base-exchange and the cytidine pathway are localized to microsomal membranes. Parallel in vivo studies showed that after the intracranial injection of L-[G-3H]serine, the specific activity of phosphatidylserine was greater in the microsomal fractions than in the mitochondrial fraction, whereas the opposite was true for phosphatidylethanolamine. When L-[U-14C]serine and [1-3H]ethanolamine were simultaneously injected, the 14C/3H ratio in mitochondrial phosphatidylethanolamine was 10 times that in microsomal phosphatidylethanolamine. The results demonstrate that serine is incorporated into the base moiety of phosphatidylethanolamine primarily through the decarboxylation of phosphatidylserine in brain mitochondria. A minimal value of 7% for the contribution of phosphatidylserine decarboxylase to whole-brain phosphatidylethanolamine synthesis can be estimated from the in vivo data.

Section: Properties and Localization Of Phosphatidylserine Decarboxylsupporting

confidence: 89%

“…The phosphatidylethanolamine band was then scraped and eluted. The purified phospholipids were hydrolyzed with phospholipase C as described by Yeung and Kuksis (1974). The completeness of hydrolysis was monitored by thin-layer chromatography.…”

Section: Lipid Analysismentioning

confidence: 99%

The Role of Phosphatidylserine Decarboxylase in Brain Phospholipid Metabolism

Butler

Morell

1983

“…Phospholipids were eluted from silica gel by three successive cycles of agitation, as above, with either chloroform/methanol/water (7:7: 1 by volume) for PC and PE or chloroform/methanol/water/acetic acid (50: 39: 10: 1 by volume) for PI and PS. Phospholipid classes were then converted to DG with a procedure adapted from that of Yeung and Kuksis (1974) and Kito et al (1985), which involves digestion with phospholipase in a two-phase (aqueous/diethyl ether) system. The incubation involved dispersion of lipids eluted from the silica gel in 200 p1 of 0.1 M Tris-HC1 (pH 7.4) containing 10 mM CaClz and 30 mM H3B03 (to reduce 2,3-acyl migration).…”

Section: Extraction and Quantification Of Lipidsmentioning

confidence: 99%

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn‐1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

Allen

Gammon

Ousley

et al. 1992

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.

“…To determine incorporation specific for the base moiety, phospholipids were separated as described above and the individual phospholipid classes eluted from the gel with chloroform/methanol/water/acetic acid (50:39: 1O:l) (Holub and Kuksis, 1971). The individual phospholipid classes were hydrolyzed with phospholipase C, as described by Yeung and Kuksis (1974). After hydrolysis, the solvent was evaporated with nitrogen and the residue resuspended in 0.7 ml of water, plus 2.8 ml of chloroform/methanollconcentrated hydrochloric acid (100:200:2).…”

Section: Radioactive Labeling In Vivo and Subsequent Lipid Analysismentioning

confidence: 99%

Developmental Pattern for Phosphatidylserine Decarboxylase in Rat Brain

White

Toews

Morell

1986

In adult rats, a significant portion of brain ethanolamine glycerophospholipids are synthesized by a pathway involving phosphatidylserine decarboxylase, a mitochondrial enzyme. We have now examined whether this enzyme plays a particularly prominent role during development. Activities for both phosphatidylserine decarboxylase and succinate dehydrogenase (another mitochondrial enzyme) were determined in brain homogenates from rats 5 days of age to adulthood. Succinate dehydrogenase activity, expressed on a per unit brain protein basis, increased markedly during development. This pattern has been reported previously and is as expected from the postnatal increase in oxidative metabolism. In contrast, phosphatidylserine decarboxylase activity decreased 40% from 5 to 30 days of age. The apparent Km for brain phosphatidylserine decarboxylase was 85 microM in both young (8- and 20-day-old) and adult animals. Parallel studies in vivo were carried out to determine the contribution of the phosphatidylserine decarboxylase pathway, relative to pathways utilizing ethanolamine directly, to the synthesis of brain ethanolamine glycerophospholipids. Animals were injected intracranially with a mixture of L-[G-3H]serine and [2-14C]ethanolamine and incorporation into the base moieties of the phospholipids determined. The 3H/14C ratio of ethanolamine glycerophospholipids decreased about 50% during development. Our studies in vitro and in vivo both suggest that phosphatidylserine decarboxylase plays a significant role in the synthesis of brain ethanolamine glycerophospholipids at all ages, although it is relatively more prominent early in development.