Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain

Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross-reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol.     

Packing and electrostatic behavior of sn-2-docosahexaenoyl and -arachidonoyl phosphoglycerides

Mammalian synaptic membranes appear to contain high proportions of specific, sn-1-stearoyl-2-docosahexaenoyl- and sn-1-stearoyl-2-arachidonoyl phosphoglycerides, but the structural significance of this is unclear. Here we used a standardized approach to compare the properties of homogeneous monolayers of the corresponding phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidic acids with those of control monolayers of sn-1-stearoyl-2-oleoyl- and sn-1-palmitoyl-2-oleoyl phosphoglycerides. Major findings were: 1), that the presence of an sn-2-docosahexaenoyl group or an sn-2-arachidonoyl group increases the molecular areas of phosphoglycerides by 3.8 A(2) (7%) relative to the presence of an sn-2-oleoyl group; 2), that the phosphorylcholine headgroup independently increases molecular areas by a larger amount, 7.1 A(2) (13%); and 3), that the dipole moments of species having an arachidonoyl moiety or an oleoyl moiety are 83 mD (19%) higher than those of comparable docosahexaenoic acid-containing phosphoglycerides. These and other results provide new information about the molecular packing properties of polyenoic phosphoglycerides and raise important questions about the role of these phosphoglycerides in synapses.     

A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.     

Affinity of phosphatidylcholine molecular species for the bovine phosphatidylcholine and phosphatidylinositol transfer proteins. Properties of the sn-1 and sn-2 acyl binding sites

Both the phosphatidylcholine transfer protein (PC-TP) and the phosphatidylinositol transfer protein (PI-TP) act as carriers of phosphatidylcholine (PC) molecules between membranes. To study the structure of the acyl binding sites of these proteins, the affinity of 32 distinct natural and related PC molecular species was determined by using a previously developed fluorometric competition assay. Marked differences in affinity between species were observed with both proteins. Affinity vs lipid hydrophobicity (determined by reverse-phase HPLC) plots displayed a well-defined maximum indicating that the acyl chain hydrophobicity is an important determinant of binding of a phospholipid molecule by these transfer proteins. However, besides the overall lipid hydrophobicity, steric properties of the individual acyl chains contribute considerably to the affinity, and PC-TP and PI-TP respond differently to modifications of the acyl chain structure. The affinity of PC-TP increased steadily with increasing unsaturation of the sn-2 acyl moiety, resulting in high affinity for species containing four and six double bonds in the sn-2 chain, whereas the affinity of PI-TP first increased up to two to three double bonds and then declined. These data, as well as the distinct effects of sn-2 chain double bond position and bromination, indicate that the sn-2 acyl chain binding sites of the two proteins are structurally quite different. The sn-1 acyl binding sites are dissimilar as well, since variation of the length of saturated sn-1 chain affected the affinity differently. The data are discussed in terms of the structural organization of the sn-1 and sn-2 acyl binding sites of PC-TP and PI-TP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain

We have studied the properties of the fatty acyl binding sites of the phosphatidylinositol transfer protein (PI-TP) from bovine brain, by measuring the binding and transfer of pyrenylacyl-containing phosphatidylinositol (PyrPI) species and pyrenylacyl-containing phosphatidylcholine (PyrPC) species as a function of the acyl chain length. The PyrPI species carried a pyrene-labeled acyl chain of variable length in the sn-2 position and either palmitic acid [C(16)], palmitoleic acid [C(16:1)], or stearic acid [C(18:1)] in the sn-1 position. Binding and transfer of the PI species increased in the order C(18) less than C(16) less than C(16:1), with a distinct preference for those species that carry a pyrenyloctanoyl [Pyr(8)] or a pyrenyldecanoyl [Pyr(10)] chain. The PyrPC species studied consisted of two sets of positional isomers: one set contained a pyrenylacyl chain of variable length and a C(16) chain, and the other set contained an unlabeled chain of variable length and a Pyr(10) chain. The binding and transfer experiments showed that PI-TP discriminates between positional isomers with a preference for the species with a pyrenylacyl chain in the sn-1 position. This discrimination is interpreted to indicate that separate binding sites exist for the sn-1 and sn-2 acyl chains. From the binding and transfer profiles it is apparent that the binding sites differ in their preference for a particular acyl chain length. The binding and transfer vs chain length profiles were quite similar for C(16)Pyr(x)PC and C(16)Pyr(x)PI species, suggesting that the sn-2 acyl chains of PI and PC share a common binding site in PI-TP.     

Conformationally constrained analogues of diacylglycerol (DAG). Effect on protein kinase C (PK-C) binding by the isosteric replacement of sn-1 and sn-2 esters in DAG-lactones

In order to determine the importance of the two ester pharmacophores in high affinity, conformationally constrained DAG-lactones (Lac-1-5) as PK-C ligands, we have independently replaced the sn-1 and sn-2 carbonyl esters in these compounds by ketone (2, 10, 11), amide (3, 25-28), and hydroxyl (12, 13) isosteres. Although the ketone analogue of the sn-1 ester (2) exhibited comparable activity to the parent Lac-1 when taking into account the difference in lipophilicities, the other isosteres were significantly poorer PK-C alpha ligands compared to the parent DAG-lactones. This study demonstrates that the ester functionality in DAG-lactone plays an important role in the ligand's capacity to form a strong hydrogen bond with Gly253 at the active site. The discrete K(i) analysis from the sn-1 and sn-2 isosteres further confirms that the DAG-lactones bind preferentially to the C1-domain in the sn-2 binding mode, as previously suggested.     

The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities.

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.     

Synthesis of sn-1 functionalized phospholipids as substrates for secretory phospholipase A2

Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl-substituents attached to the sn-1 position of the glycerol backbone. The synthesis of phospholipids 1 and 2 is based upon the construction of a key aldehyde intermediate 3 which locks the stereochemistry in the sn-2 position of the final phospholipids. The aldehyde functionality serves as the site for insertion of the allyl-substituents by a zinc mediated allylation. Small unilamellar liposomes composed of phospholipids 1 and 2 were subjected to sPLA2 activity measurements. Our results show that only phospholipid 1 is hydrolyzed by the enzyme. Molecular dynamics simulations revealed that the lack of hydrolysis of phospholipid 2 is due to steric hindrance caused by the bulky side chain of the substrate allowing only limited access of water molecules to the active site.     

Structured triacylglycerol containing medium-chain fatty acids in sn-1(3) facilitates the absorption of dietary long-chain fatty acids in rats

A study was carried out to examine if the positional distribution of medium chain fatty acids (MCF) in triacylglycerol influences dietary fat absorption in rats. Two types of structure-specific fats, one predominantly composed of MCF in sn-1(3) and iinoleic acid in sn-2 [sn1(3)MCF-structured] and the others of MCF in sn-2 and linoleic acid in sn-1(3) [sn-2MCF-structured], were initially prepared, and the two structure-specific fats were interesterified and designated as sn-1(3)MCF-interesterified and sn-2MCF-interesterified. Synthetic fat was mixed with an equal amount of cocoa butter (103 g/kg of diet) and was supplemented to the AIN93G-based diet. Rats were fed on the diets for 4 wk. Long-chain saturated fatty acids were the predominant fatty acids excreted into the feces, and the positional distribution of MCF resulted in an altered fat absorption rate (%) of 81.8, 82.5, 84.2 and 86.3 for the rats fed on the diets containing sn-2MCF-structured, sn-1(3)MCF-interesterified, sn-2MCF-interesterified and sn-1(3)MCF-structured fats, respectively. The proportion of MCF in the serum, liver and adipose tissue triacylglycerols was not affected by the MCF distribution of the dietary fats. These results indicate that the distribution of MCF in dietary triacylglycerol is a determinant of intestinal fat absorption.     

sn-1-Acylglycerol-3-phosphate acyltransferase of Escherichia coli causes insertion of cis-11 eicosenoic acid into the sn-2 position of transgenic rapeseed oil

The plsC gene of Escherichia coli encoding sn-1-acylglycerol-3-phosphate acyltransferase was modified by inserting an endoplasmic reticulum retrieval signal to its 3 end and introduced into rapeseed (Brassica napus L.) plants under the control of a napin promotor. In developing seeds from transgenic plants an sn-1-acylglycerol-3-phosphate acyltransferase activity was detectable which showed substrate specificities typical of the E. coli enzyme. Moreover, seed oil from the transformants unlike that from untransformed plants contained substantial amounts of triacylglycerol species esterified with very-long-chain fatty acids at each glycerol position. Analysis of fatty acids at the sn-2 position of triacylglycerol showed hardly any very-long-chain fatty acids in untransformed plants, but in certain transformants these fatty acids were present, namely about 4% erucic acid and 9% eicosenoic acid. These data demonstrate that the bacterial acyltransferase can function in developing rapeseed and alters the stereochemical composition of transgenic rape seed oil by directing very-long-chain fatty acids, especially cis-11 eicosenoic acid, to its sn-2 position.     

Evidence that remodeling of the fatty acids of phosphatidylcholine is regulated in isolated rat hepatocytes and involves both the sn-1 and sn-2 positions

The remodeling of the fatty acyl moieties of phosphatidylcholine (PC) has been studied in choline-deficient and choline-supplemented hepatocytes prepared from a choline-deficient rat. Choline-deficient hepatocytes were prelabeled with [Me-3H]choline for 30 min and subsequently incubated for up to 12 h in the presence or absence of choline. Analysis of the molecular species of PC from choline-deficient cells showed that, at the end of the pulse, approx. 75% of the label was incorporated into palmitate-containing species and only approx. 16% of the labeled species contained stearate. During the chase period there was a redistribution of label and after 12 h approx. 56% of the total radioactivity was associated with palmitate containing species and 37% was recovered in stearate-containing species. A similar distribution of radioactivity was observed in choline-supplemented cells. Measurement of the specific radioactivity of the major molecular species of PC was consistent with a precursor-product relationship between palmitate-containing species and stearate-containing species with arachidonate or linoleate on the sn-2 position. A model is presented which takes into account remodeling of both the sn-1 and sn-2 positions of PC.     

Human plasma lecithin-cholesterol acyltransferase. Inhibition of the phospholipase A2-like activity by sn-2-difluoroketone phosphatidylcholine analogues

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the sn-2-fatty acid of lecithin to cholesterol, forming lysolecithin and cholesteryl ester. We have recently proposed a covalent catalytic mechanism for LCAT in which lecithin cleavage proceeds via the formation of a transition state tetrahedral adduct between the oxygen atom of the catalytic serine residue and the sn-2-carbonyl carbon atom of the substrate (Jauhiainen, M., Ridgway, N.D., and Dolphin, P.J. (1987) Biochim. Biophys. Acta 918, 175-188). This proposal is evaluated here by use of nonhydrolyzable sn-2-difluoroketone phosphatidylcholine analogues, known to inhibit calcium-dependent phospholipase A2. These compounds inhibited the calcium-independent phospholipase A2 activity of LCAT in a time and concentration dependent manner. The most potent analogues had a 100-fold higher affinity for the enzyme than the substrate, lecithin, when present within lecithin/apoA-I proteoliposomes. The inhibition was dependent upon the presence of a difluoromethylene group alpha to the sn-2-carbonyl carbon of the analogues. The inhibition is attributed to the formation of a tetrahedral adduct between the catalytic serine residue of LCAT and the sn-2-carbonyl carbon atom of the analogues which is stabilized by the electronegative fluorine atoms present upon the carbon atom alpha to the carbonyl carbon. This adduct mimics that proposed by us to occur during lecithin cleavage by LCAT, and the data substantiate the existence of this transition state adduct prior to the release of lysolecithin and formation of a fatty acylserine oxyester of the enzyme.     

Altered positional specificity of human plasma lecithin-cholesterol acyltransferase in the presence of sn-2 arachidonoyl phosphatidyl cholines. Mechanism of formation of saturated cholesteryl esters.

The positional specificity of purified human lecithin-cholesterol acyltransferase (LCAT) was studied by analyzing the labeled cholesteryl ester (CE) species formed in the presence of proteoliposome substrates containing mixed chain phosphatidylcholine (PC) species, labeled cholesterol and apoprotein A-I. Whereas over 90% of the acyl groups used for CE synthesis were derived from the sn-2 position of most of the naturally occurring PC substrates, about 75% of the CE species formed in the presence of sn-1-myristoyl 2-arachidonoyl PC, sn-1-palmitoyl-2-arachidonoyl (PAPC) and sn-1-palmitoyl 2-docosahexaenoyl PC were derived from the sn-1-position. On the other hand, rat LCAT utilized mostly sn-2-acyl group from either PAPC or from sn-1-palmitoyl 2-linoleoyl PC. The positional specificity of the human enzyme was not affected by the alteration in the matrix fluidity, type of the apoprotein activator used, or by the free cholesterol/PC ratio in the substrate. These results show that the positional specificity of human plasma LCAT is altered in the presence of sn-2-arachidonoyl PC, or sn-2-docosahexaenoyl PC, probably due to steric restrictions at the active site, and this may account for the formation of disproportionately high concentrations of saturated CE, and low concentrations of long-chain polyunsaturated CE in human plasma, relative to the composition of sn-2-acyl groups in plasma PC.     

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.     

Effects of various numbers and positions of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the chain-melting temperature

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.     

sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.     

Tributyrin specifically induces a lipase with a preference for the sn-2 position of triglyceride in Geotrichum sp. FO401B

The extracellular Lipases A and C produced by Geotrichum sp. FO401B have a preference for the sn-1,3 and sn-2 positions of triglyceride, respectively. Total production of these lipases was increased by plant oils and tributyrin. Butyl Toyopearl column chromatography demonstrated that only Lipase C was produced in the presence of tributyrin. Lipase C hydrolysed natural fats except sardine oil preferentially at the sn-2 position, but it showed little stereoselectivity for triolein.     

Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm

Brassica oleracea accessions possess traits that would be useful in commercial Brassica crops. These traits can be studied more effectively through the production of doubled haploid plants. Nineteen B. oleracea accessions from several subspecies possessing significant sn-2 erucic acid were screened for suitability for microspore culture using techniques well established for Brassica. Fifteen of the 19 accessions produced embryos. Genotypic differences were observed with embryogenesis ranging from 0 to 3000 embryos/100 buds. Embryogenesis was improved for two of four accessions by initiating cultures in NLN medium with 17% sucrose, then reducing sucrose to 10% after 48 h. An increase in embryogenesis for the same two accessions was observed when microspores were cultured at a density of 100 000/ml rather than 50 000 microspores/ml. A culture temperature of 32 °C for 48 h was beneficial for three of the four accessions when compared to a longer incubation period (72 h) or a higher temperature (35 °C). One accession line, Bo-1, was found to produce microspore-derived embryos which contained triacylglycerols with significant proportions of erucic acid at the sn-2 position. This revised version was published online in June 2006 with corrections to the Cover Date.