Diacylglycerol lipase and kinase activities in rat brain microvessels

Diacylglycerols can accumulate transiently in intact cells as a consequence of the degradation of phosphatidylinositol by phospholipase C, but little information is available concerning their metabolic fate in the vascular endothelium. Diacylglycerol lipase and kinase activities were measured in rat brain microvessel preparations. Lipase activity, measured by the release of free fatty acids, was much greater at pH 4.5 than at pH 7. The acid lipase was predominantly particulate and likely originated in lysosomes, whereas the neutral lipase was mainly soluble. The fatty acid at the sn-1 position of the diacylglycerol substrate was hydrolyzed faster than that at the sn-2 position at both pH 4.5 and 7. The 2-monoacylglycerol accumulated at pH 4.5 but not at 7 due to the presence of a monoacylglycerol lipase activity with a neutral pH optimum. The formation of phosphatidic acid (kinase activity) was also measured in microvessels. When lipase and kinase activities were measured simultaneously, the formation of phosphatidic acid from a 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol substrate was 4-fold greater than the release of fatty acid (oleate) from the sn-2 position. Introduction of arachidonic acid to the sn-2 position of the diacylglycerol substrate increased kinase activity but reduced lipase activity. The release of fatty acids from the sn-2 position of phosphatidic acid could not be detected.     

Diacylglycerol metabolism and arachidonic acid release in human fetal membranes and decidua vera

Diacylglycerol lipase activity has been demonstrated in human fetal membranes and decidua vera tissues. The specific activity of the enzyme is highest in the microsomal fraction of decidua vera tissue. The acylester bond at the sn-1 position of 1,2-diacyl-sn-glycerol is hydrolyzed followed by release of the fatty acid at the sn-2 position. The diacylglycerol lipase activity present in the microsomal fraction of decidua vera tissue hydrolyzes preferentially a diacylglycerol containing an arachidonoyl group in the sn-2 position. Monoacylglycerol lipase activity was also demonstrated in these tissues. The specific activity of monoacylglycerol lipase was significantly greater than that of diacylglycerol lipase and catalyzed preferentially the hydrolysis of monoacylglycerols containing an arachidonyl group in the sn-2 position. Based on the subcellular distribution and the differential effects of various inhibitors, we suggest that the monoacylglycerol lipase and diacylglycerol lipase in decidua vera tissue are 2 distinct enzymes. Diacylglycerol kinase specific activity was examined also and was found to be 4-5 times greater in amnion than in either chorion laeve or decidua vera. The importance of diacylglycerol metabolism in the mechanism of arachidonic acid release and prostaglandin biosynthesis is discussed.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

The activity and properties of an acidic triacylglycerol lipase from adult and fetal rat lung

Triacylglycerol lipase with maximal activity at pH 5 was present in adult and fetal lung. The activity was inhibited by serum concentrations used to measure lipoprotein lipase and by 0.5 M NaCl. The activity in homogenates from fetal lung was about 40% of the activity in adult lung homogenates. The activity increased to 80% of the adult levels during the first 24–48 h following birth. Acidic triacylglycerol lipase was present in all subcellular fractions from adult lung. However, the major amount of activity appeared to be associated with lysosomes. Fetal lung contained significantly more activity in the cytosolic fraction compared to the adult. The reaction produced free fatty acids (65%), 1,2(2,3)-diacylglycerol (22%) and 2-monoacylglycerol (12%). Minimal amounts of 1,3-diacylglycerol and 1(3)-monoacylglycerol were formed. Diacylglycerol lipase and monoacylglycerol hydrolase activities at pH 5 were independently determined and both were higher than the triacylglycerol lipase activity. The subcellular distribution of diacylglycerol lipase and monoacylglycerol hydrolase differed from that of triacylglycerol lipase. Overall, the results indicated that the lung has considerable intracellular lipase activity and therefore could readily hydrolyze intracellular triacylglycerol to free fatty acids. The reaction also produced significant amounts of 1,2-diacylglycerol which suggests that triacylglycerol could be a direct source of diacylglycerol for phospholipid synthesis.     

Arachidonic acid release from diacylglycerol in human neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an intracellular pool to plasma membrane upon cell activation

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.     

Generation of lysoglyceroglycolipids in the cyanobacterium, Phormidium tenue.

Two kinds of lysoglycolipids, monogalactosyl 1-monoacylglycerol and digalactosyl 1-monoacylglycerol were generated in the cyanobacterium, Phormidium tenue, when it was stored at -20 degrees for more than 1 month. By comparison of the compositions of fatty acid residues between monogalactosyl 1-monoacylglycerol and monogalactosyl diacylglycerol, digalactosyl 1-monoacylglycerol and digalactosyl diacylglycerol, respectively, the 1-monoacylgalactolipids were presumed to be formed by regioselective deacylation at the sn-1 position with subsequent acyl-group migration from the sn-2 to the sn-1 position. In contrast, 1-monoacyl derivatives of sulphoquinovosyl diacylglycerol and phosphatidylglycerol, the other membrane lipids contained in cyanobacteria, were not formed.     

Diacylglycerol hydrolysis in rat liver lysosomes

The matrix of rat liver lysosomes exhibits high hydrolytic activity towards 1,2-diacylglycerol with an optimum at pH 4.0. The lipolytic reaction follows Michaelis-Menten kinetics (apparent Vmax 470 nmol hydrolysed/min per mg protein; apparent Km 71 microM 1,2-dioleoylglycerol). Formation of 1- and 2-monooleoylglycerols indicates an initial attack at both the primary and secondary ester bonds. The lysosomal matrix also catalyses (re)acylation reactions, i.e. the formation of 1,2-diacylglycerol from 2-monoacylglycerol and free fatty acid. However, (re)acylation proceeds at a far lower rate than deacylation of diacylglycerol. Lysosomal diacylglycerol hydrolysis is sensitive towards non-ionic detergents, cationic amphiphilic drugs and the lipase inhibitor RHC 80267.     

Purification and characterization of intracellular lipase from the polyunsaturated fatty acid-producing fungus Mortierella alliacea

Previous studies on an arachidonic acid-producing fungus, Mortierella alliacea YN-15, suggested that its intracellular lipase plays an important role in the metabolism of exogenous and storage lipids. The lipase purified in this study through acetone precipitation and three-step chromatography was estimated to be about 11 kDa in size by SDS-PAGE and mass spectrometry, and it tended to form large aggregates in aqueous solution. The purified lipase retained its activity over wide ranges of pH (2-12) and temperature (20-80 °C). Its activity was enhanced by the Ca(2+) ion and reduced by some heavy metal ions, such as Zn(2+) and Hg(2+), and diethylpyrocarbonate. Among the various substrates tested, monoacylglycerols containing long-chain unsaturated fatty acids and phosphatidylcholine were preferentially hydrolyzed over triacylglycerols and fatty acid methyl esters. The lipase strongly hydrolyzed the sn-1/3 ester bonds and weakly hydrolyzed the sn-2 ester bonds of triolein, and it also catalyzed the acylglycerol synthesis reaction in a solvent-free two-phase system. The results indicate that triacylglycerol may be formed via 2-monoacylglycerol. Thus, the highly stable M. alliacea lipase may be useful for the synthesis of structured lipids, particularly acylglycerols containing functional unsaturated fatty acids at the sn-2 position.     

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.     

Characterization of a partially purified diacylglycerol lipase from bovine aorta

A partially-purified diacylglycerol (DG) lipase from bovine aorta has been characterized with respect to the effects of lipid metabolites and two lipase inhibitors, phenylboronic acid and tetrahydrolipstatin (THL). DG lipase activity was determined by the hydrolysis of the sn-1 position of 1-[1-⁴C]palmitoyl-2-oleoyl-sn-glycerol. The products of the lipase reaction, 2-monoacylglycerol (2-monoolein) and non-esterified fatty acids (oleate, arachidonate) produced a concentration-dependent (20–200 μM) inhibition of DG lipase activity. Oleoyl-CoA and dioleoylphosphatidic acid also inhibited aortic DG lipase activity, but lysophosphatidylcholine had little or no effect. The inhibition of aortic DG lipase by phenylboronic acid was competitive, with a K_i of approx. 4 mM. THL was a very potent inhibitor of aortic DG lipase; the concentration required for inhibition to 50% of control was 2–6 nM. THL was a very potent inhibitor of concentration of substrate in the assay was increased. Attempts to identify the aortic DG lipase by covalent-labelling with [¹⁴C]THL were unsuccessful. Immunoblotting experiments revealed that hormone-sensitive triacylglycerol lipase (HSL) could not be detected in bovine aorta.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence

Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements.     

Triglyceride metabolism in 3T3-L1 cells. An in vivo 13C NMR study.

13C nuclear magnetic resonance spectroscopy has been used to study triglyceride metabolism in 3T3-L1 cells incubated with [1-13/14C] acetate, myristate, palmitate, stearate, or oleate. Labeled cells embedded in agarose filaments were perfused in a specially fitted NMR tube within the spectrometer magnet. Incubation of 3T3-L1 cells with a specific fatty acid enriched the cellular triglycerides with that fatty acid; the NMR signal observed in the carbonyl region of the cell spectrum was due in large part to that fatty acid. NMR data demonstrated that cellular enzymes preferentially esterified saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position. cellular triglyceride hydrolysis by hormone-sensitive lipase was monitored by measuring the decrease in the integrated intensities of resonances arising from fatty acyl carbonyls esterified at glycerol carbons sn-1,3 and sn-2. Under basal conditions, the time courses were first-order, and the average rates were 0.14% of signal/min at both carbonyl positions. Under isoproterenol stimulated conditions, these rates were still first-order and increased 6.4-fold at the sn-1,3 position and 2.4-fold at the sn-2 position. The observation that the hydrolysis time courses were first-order suggested that only a small amount of cellular triglyceride was available to hormone-sensitive lipase, supporting the view that lipolytic enzymes operate at lipid surfaces where only small amounts of neutral lipid may be soluble. Attempts to correlate the measured rates with the rates of hydrolysis at the sn-1,3 and sn-2 positions were hindered by the fact that the chemical shifts of the carbonyl carbons of the diglyceride hydrolysis product did not overlie those of the triglyceride. Analysis of hydrolysis kinetics revealed that hormone-sensitive lipase exhibited little preference for a particular esterified fatty acid under basal conditions; however, under stimulated conditions, the enzyme exhibited a preference for certain triglyceride species.     

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells

Summary The effect of a reduction in protein kinase C activity on the metabolism of exogenous [³H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [³H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [³H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.Abbreviations IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - MG monoacylglycerol - PL phospholipid(s) - diC8 dioctanoylglycerol - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - monoC8 monooctanoylglycerol - PS phosphatidylserine - PDBu phorbol 12,13-dibutyrate     

Regulation of diacylglycerol kinase in the transition from quiescence to proliferation in Dictyostelium discoideum

Diacylglycerol kinase and phosphatidylinositol kinase were examined in stationary phase D. discoideum amoeba induced to synchronously proliferate by dilution into fresh medium. Membrane bound diacylglycerol kinase activity showed a rapid and transitory 3-5 fold increase in the preproliferative interphase while phosphatidylinositol kinase activity was kept quite constant during the same period. The changes in diacylglycerol kinase activity seem to be due to a translocation of the enzyme from the soluble to the particulate cell compartments.     

Expression and characterization of diacylglycerol acyltransferase from Arabidopsis thaliana in insect cell cultures

Diacylglycerol acyltransferase (DGAT) catalyses the acylation of the sn-3 hydroxy group of sn-1,2-diacylglycerol using acyl-CoA. The gene encoding DGAT from Arabidopsis thaliana has been cloned and the function of the enzyme proved by expression of the coding sequence using a bacculovirus expression system in insect cell cultures. The expressed protein catalysed the synthesis of [(14)C]triacylglycerol from [(14)C]diacylglycerol and oleoyl-CoA. The heterologously expressed DGAT activity was found mostly associated with the 100000 g pellet. The optimum activity was achieved at a neutral pH, in the presence of Mg2+, and at an optimum oleoyl-CoA concentration of 20 microM. The DGAT used the substrates palmitoyl-CoA and oleoyl-CoA equally effectively. In these experiments, the inclusion of recombinant acyl-CoA binding protein had a relatively small effect upon DGAT activity.     

Synthesis of sn-1,2-diacylglycerols by monoacylglycerol acyltransferase from Manduca sexta fat body

The pathway for the synthesis of sn-1,2-diacylglycerol stimulated by the action of adipokinetic hormone (AKH) in the insect fat body is unknown. Previous results from this laboratory suggested that the hydrolysis of stored triacylglycerol to sn-2-monoacylglycerol followed by the stereospecific acylation of sn-2-monoacylglycerol catalyzed by a monoacylglycerol-acyltransferase (MGAT) could be the major route of AKH-stimulated sn-1,2-diacylglycerol synthesis. Thus, MGAT might represent a key enzyme of this pathway. In this study we characterized the MGAT activity from the Manduca sexta fat body. The activity, which was assayed by acylation of 2-monoolein using radioactive labeled palmitoyl-CoA, was found to be primarily a microsomal enzyme. The products of the acylation of 2-monoolein were 1,2-diacylglycerol (40–50%), 1,3-diacylglycerol (20–30%), and triacylglycerol (30–40%). The presence of triacylglycerol as a product revealed the presence of diacylglycerol-acyltransferase activity in the fat body microsomes. The pH optimum of MGAT activity was 7.0, and the dependence of the activity on the concentration of 2-monoolein showed saturation kinetics. An endogenous MGAT activity, which represented 20% of the maximal activity observed with added substrate, was detected. Optimal concentrations of palmitoyl-CoA ranged between 0.10–0.20 mM. The specific activity of MGAT, measured under optimal conditions, was about 0.6 nmol DG formed/min-mg protein. MGAT activity was greatest with 2-monoolein, and lower activity was observed when a saturated 2-monoacylglycerol was employed. The activity observed with sn-1-monoacylglycerol was lower than that observed with sn-2-monoacylglycerol. AKH did not stimulate MGAT activity, suggesting that either the enzyme is not under hormonal regulation or the monoacylglycerol pathway is not involved in the AKH-stimulated production of sn-1,2-diacylglycerol in the M. sexta fat body. © 1996 Wiley-Liss, Inc.     

Apparent convergence (at 2-monoacylglycerol level) of phosphatidic acid and 2-monoacylglycerol pathways of synthesis of chylomicron triacylglycerols

Dietary fats are converted into chylomicron triacylglycerols via the 2-monoacylglycerol and phosphatidic acid pathways of acylglycerol formation. In view of the known positional and fatty acid specificity of the acyltransferases, the triacylglycerol structures resulting from the two pathways would be expected to differ, but this has not been demonstrated. We have performed stereospecific analyses on the chylomicron triacylglycerols from rats fed menhaden oil and the corresponding fatty acid alkyl esters, which would be expected to be assimilated via the monoacylglycerol and the phosphatidic acid pathways, respectively. The results show a remarkable similarity between the two triacylglycerol types in the fatty acid composition of the sn-1 and sn-3 positions, along with marked differences in the composition of the sn-2 positions. The triacylglycerols from rats fed oil retained about 85% of the original fatty acids in the sn-2 position, including a high proportion of the long chain polyunsaturates (e.g., 5-7% 20:5 and 4-5% 22:6). The triacylglycerols from rats fed the alkyl ester contained large amounts of endogenous fatty acids in the sn-2 position (e.g., 18% 16:1, 14% 18:1, 14% 18:2, and 2.5% 20:4), which approximated the composition of the sn-2 position of the presumed phosphatidic acid intermediates. The sn-1 position contained a much higher proportion of polyunsatured fatty acids (e.g., 12-13% 20:5, 5-6% 22:6) than the sn-2 position (e.g. 2-3% 20:5, 0-0.6% 22:6) of triacylglycerols from rats fed the ester. We conclude that the chylomicron triacylglycerols arising via the 2-monoacylglycerol and the phosphatidic acid pathways differ mainly in the composition of the fatty acids in the sn-2 position. The similarity in the acids of the sn-1 and sn-3 positions of the chylomicron triacylglycerols from rats fed oil or ester is consistent with a hydrolysis of the acylglycerol products of the phosphatidic acid pathway to 2-monoacylglycerols prior to reconversion to triacylglycerols via the monoacylglycerol pathway and secretion as chylomicrons.