Phosphatidic acid metabolism in rat liver cell nuclei

… et Biophysica Acta (BBA …, 1989

Thiophosphatidic acid (|,2-diaey|-sn-glycero-3-phosphorothioate; thioPA) was chemically synthesized from egg phosphatidyleholine-derived 1,2-diacy|glycerol and PSC| 3 and tested for its effects on enzymes which utilize phosphatidic acid (PA) in phospholipid biosynthesis. The compound was not a substrate for rat liver cytosolic PA phosphatase and strongly inhibited this enzyme activity. ThioPA was also a potent inhibitor of purified membrane-associated PA phosphatase from Saccharomyces cerevisiae in a competitive manner and exhibited an apparent Ki=60 pM. In contrast, purified CDPdiacylglycerol synthase (PA:CTP cytidylyltransferase) from this organism was able to convert thioPA to CDP-diacy|glycerol. The apparent V,,ia,, for thioPA was 7-fold |ower than that for PA, whereas the apparent K m for thioPA (70 /zM) was 4-fold lower than ~hat for PA. Calculation of the specificity constant (Vmax//Km) demonstrated that PA was the preferred substrate. These properties of thioPA indicate that this substance may prove useful in studies of phospholipid metabolism and function.

Biology of the Cell, 2004

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCß1 having a role in the onset of DNA synthesis and PC-PLCc1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelinlinked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance

FEBS Letters, 2004

Phospholipase C (PLC) was purified from the membrane-depleted rat liver nuclei. About 60% of the total PLCactivity corresponded to b 1b isoform, 30% to PLC-c 1 and less than 10% to PLC-d 1 . PLC-b 1b and -c 1 were found in the nuclear matrix, while PLC-d 1 was detected in the chromatin. Two peaks of an increase in the total PLC-activity were detected occurring at 6 and 20 h after partial hepatectomy. An early increase in PLC-b 1b activity in the nuclear matrix was associated with serine phosphorylation of the enzyme, while the later increase paralleled the increase in the amount of protein. The increase in the PLC-c 1 activity measured at 6 and 20 h after partial hepatectomy was associated with tyrosine phosphorylation of the enzyme. The activity of PLC-d 1 and the amount of the protein found in the chromatin was increased only at 20 h after partial hepatectomy.

Journal of Biological Chemistry, 1973

The soluble fraction from the plasma membranes was prepared by treatment with heparin as described earlier (8). Lipolytic activity was measured using 50 pM of each lipid added 7985 This is an Open Access article under the CC BY license.

Archives of Biochemistry and Biophysics, 1997

Increasing observations indicate that both L-FABP 3 and I-FABP enhance the esterification of fatty acids The effect of fatty acid binding proteins (FABPs) on into triglycerides and phospholipids. Expression of Ltwo key steps of microsomal phosphatidic acid forma-FABP in transfected fibroblasts stimulated fatty acid tion was examined. Rat liver microsomes were puriuptake and oleic acid esterification into neutral lipids fied by size-exclusion chromatography to remove enand even more so into phospholipids (1-4). This redogenous cytosolic fatty acid and fatty acyl-CoA binding proteins while recombinant FABPs were used to sulted in a net increase in cellular phospholipid mass avoid cross-contamination with such proteins from (3, 5). In contrast, expression of I-FABP in transfected native tissue. Neither rat liver (L-FABP) nor rat intesfibroblasts did not enhance fatty acid uptake but, nevtinal fatty acid binding protein (I-FABP) stimulated ertheless, resulted in selective incorporation of oleic liver microsomal fatty acyl-CoA synthase. In contrast, acid into neutral lipids such as triglycerides and L-FABP and I-FABP enhanced microsomal convercholesteryl esters at the expense of incorporation into sion of [ 14 C]oleoyl-CoA and glycerol 3-phosphate to phospholipids (1, 2), such that net cellular mass of [ 14 C]phosphatidic acid by 18-and 7-fold, respectively. these neutral lipids increased (2). Furthermore, L-The mechanism for this stimulation, especially by I-FABP and I-FABP enhanced selective incorporation of FABP, is not known. However, several observations oleic acid within the phospholipid fraction, primarily presented here suggest that, like L-FABP, I-FABP into phosphatidylcholine and phosphatidylethanolammay interact with fatty acyl-CoA and thereby stimuine (1-3). Thus, both L-FABP and I-FABP stimulated late enzyme activity. First, I-FABP decreased microesterification of fatty acids into phosphatidic acid-desomal membrane-bound oleoyl-CoA. Second, oleoylrived triglycerides and phospholipid subclasses in in-CoA displaced I-FABP bound fluorescent fatty acid, tact cells. cis-parinaric acid, with K i of 5.3 mM and 1.1 sites. The two major steps leading to intracellular incorpo-Third, oleoyl-CoA decreased I-FABP tryptophan fluration of fatty acids into phosphatidic acid are the actiorescence with a K d of 4.2 mM. Fourth, oleoyl-CoA red vation of fatty acid to fatty acyl-CoA (fatty acyl-CoA shifted emission spectra of acrylodated I-FABP, a sensynthetase) and the incorporation of fatty acyl-CoA sitive marker of I-FABP interactions with ligands. In into glycerol 3-phosphate to form phosphatidic acid summary, the results demonstrate for the first time (glycerol-3-phosphate acyltransferase and lysophosthat both L-FABP and I-FABP stimulate liver microphatidate acyltransferase) [reviewed in (6)]. The longsomal phosphatidic acid formation by enhancing synchain fatty acyl-CoA synthetase is found mainly in the thesis of phosphatidate from fatty acyl-CoA and glycendoplasmic reticulum and mitochondria of mammaerol 3-phosphate. ᭧ 1997 Academic Press lian cells. The rate-limiting step in the sequential acy-Key Words: fatty acid binding protein; phosphatidic lation of glycerol 3-phosphate is that catalyzed by glycacid; microsomes; liver; intestine; fatty acyl-CoA. erol-3-phosphate acyltransferase to form monoacylglycerol phosphate. In liver this enzyme is distributed