Acyl Dihydroxyacetone Phosphate Research Articles

The Crucial Step in Ether Phospholipid Biosynthesis: Structural Basis of a Noncanonical Reaction Associated with a Peroxisomal Disorder

Ether phospholipids are essential constituents of eukaryotic cell membranes. Rhizomelic chondrodysplasia punctata type 3 is a severe peroxisomal disorder caused by inborn deficiency of alkyldihydroxyacetonephosphate synthase (ADPS). The enzyme carries out the most characteristic step in ether phospholipid biosynthesis: formation of the ether bond. The crystal structure of ADPS from Dictyostelium discoideum shows a fatty-alcohol molecule bound in a narrow hydrophobic tunnel, specific for aliphatic chains of 16 carbons. Access to the tunnel is controlled by a flexible loop and a gating helix at the protein-membrane interface. Structural and mutagenesis investigations identify a cluster of hydrophilic catalytic residues, including an essential tyrosine, possibly involved in substrate proton abstraction, and the arginine that is mutated in ADPS-deficient patients. We propose that ether bond formation might be orchestrated through a covalent imine intermediate with the flavin, accounting for the noncanonical employment of a flavin cofactor in a nonredox reaction.

Facile syntheses of acyl dihydroxyacetone phosphates and lysophosphatidic acids having different acyl groups

In this study, we report novel and simple chemical syntheses of acyl dihydroxyacetone phosphate (DHAP) and 1-acyl glycero-3-phosphate [lysophosphatidic acid (LPA)], key intermediaries in the formation of glycerolipids containing ester and ether bonds. The synthesis of acyl DHAPs involved acylating the dimethyl ketal of DHAP by acid anhydride using 4-pyrrolidinopyridine as the catalyst, and the resulting product was deketalized by HClO(4) in acetone to produce acyl DHAP. The acid anhydride was either added directly or generated in the reaction mixture from the corresponding fatty acid using dicyclohexylcarbodiimide as the condensing agent. Using these methods, a number of acyl DHAPs having short-, medium-, and long-chain saturated and unsaturated acyl groups were synthesized, with overall yields from 37% to 75%. The activities of these acyl DHAPs as substrates for guinea pig liver peroxisomal acyl DHAP:NADPH reductase and alkyl DHAP synthase were then determined. Next, starting from these acyl DHAPs, a variety of LPAs were synthesized by chemical reduction of the ketone group. Biological activities of these LPAs were determined by measuring their relative abilities to release intracellular Ca(2+) via the LPA receptor. A combined chemical-enzymatic method is also described to prepare the natural LPA from the racemic mixture.

Induction of the Peroxisomal Glycerolipid-synthesizing Enzymes during Differentiation of 3T3-L1 Adipocytes: ROLE IN TRIACYLGLYCEROL SYNTHESIS

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D-[3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.

An Animal Cell Mutant with a Deficiency in Acyl/Alkyl-dihydroxyacetone-phosphate Reductase Activity: EFFECTS ON THE BIOSYNTHESIS OF ETHER-LINKED AND DIACYL GLYCEROLIPIDS

In the accompanying paper (James, P. F., and Zoeller, R. A. (1997) J. Biol. Chem. 272, 23532-23539), we reported the isolation of a series of mutants from the fibroblast-like cell line, CHO-K1, that are deficient in the incorporation of the long chain fatty alcohol, hexadecanol, into complex lipids. All but one of these mutants, FAA. K1B, were deficient in long-chain-fatty alcohol oxidase (FAO) activity. We have further characterized this FAO+ isolate. FAA.K1B cells displayed a 40% decrease in [9,10-3H]hexadecanol uptake when compared with the parent strain. Although incorporation of hexadecanol into the phospholipid fraction was decreased by 52%, the cells accumulated label in alkylglycerol (20-fold over wild type). The increase in 1-alkylglycerol labeling corresponded to a 4-fold increase in alkylglycerol mass. Short term labeling with 32Pi showed a 45-50% decrease in overall phospholipid biosynthesis in FAA.K1B. Both diacyl- and ether-linked species were affected, suggesting a general defect in phospholipid biosynthesis. Mutant cells were able to partially compensate for the decreased biosynthesis by decreasing the turnover of the phospholipid pools. The primary lesion in FAA. K1B was identified as a 95% reduction in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Whole cell homogenates from FAA.K1B were unable to reduce either acyl-dihydroxyacetone phosphate (DHAP) or alkyl-DHAP, supporting the notion that the reduction of these two compounds is catalyzed by a single enzyme. These data suggest that the biosynthesis of diacyl phospholipids, in Chinese hamster ovary cells, begins with the acylation of dihydroxyacetone phosphate as well as glycero-3-phosphate and that the "DHAP pathway" contributes significantly to diacyl glycerolipid biosynthesis. Also, the severe reduction in acyl/alkyl-DHAP reductase activity in FAA.K1B resulted in only a moderate decrease in ether lipid biosynthesis. These latter data together with the observed increase in alkylglycerol levels support the existence of a shunt pathway that is able to partially bypass the enzymatic lesion.

Alkyl dihydroxyacetone phosphate synthase in glycosomes of Trypanosoma brucei

Alkyl-dihydroxyacetone phosphate synthase (E.C. 2.5.1.26), the key enzyme in ether phospholipid biosynthesis, was demonstrated to be present in Trypanosoma brucei. The distribution of alkyl-dihydroxyacetone phosphate synthase was found to be identical to that of dihydroxyacetone phosphate acyltransferase (E.C. 2.3.1.42), which has previously been shown to be exclusively associated with the glycosome fraction (Opperdoes, F.R. (1984) FEBS Lett. 169, 35–39). Studies with gradient purified glycosomes indicated that the formation of alkyl-dihydroxyacetone phosphate was completely dependent on the presence of acyl-dihydroxyacetone phosphate. The glycosomal alkyl-dihydroxyacetone phosphate synthase activity was characterized with respect to its pH optimum, Triton X-100 sensitivity and the dependency on the concentration of the substrates palmitoyl-dihydroxyacetone phosphate and hexadecanol. Using thin-layer chromatographic and alkaline hydrolysis procedures the reaction product was identified as alkyl-dihydroxyacetone phosphate. Alkyl-dihydroxyacetone phosphate synthase was resistant to proteolytic inactivation by trypsin in intact glycosomes but not in Triton X-100 disrupted glycosomes. It is concluded that T. brucei glycosomes contain the enzymes responsible for glycero-ether bond formation analogous to mammalian peroxisomes.

Purification of Dihydroxyacetone Phosphate Acyltransferase from Guinea Pig Liver Peroxisomes

Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), a peroxisomal enzyme which initiates the biosynthesis of glycerolipids (especially the ether-linked glycerolipids) in higher eukaryotes, has been purified by over 3250-fold from guinea pig liver. Initial stages of purification entailed isolation of liver peroxisomes by a combination of differential and density-gradient centrifugation. Dihydroxyacetone phosphate acyltransferase was solubilized from peroxisomal membranes with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate at moderate ionic strength (0.15 M NaCl). The solubilized enzyme was further purified by a regimen of size-exclusion chromatography, cation-exchange chromatography, and hydroxylapatite chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions during the purification of the enzyme, a 69-kDa protein band copurified with the enzyme activity, indicating that the monomeric enzyme may have a Mr of 69,000. This was verified by further purifying the enzyme by chromatofocusing, when a single 69-kDa band was observed on SDS-PAGE. The Mr of dihydroxyacetone phosphate acyltransferase determined by gel filtration is 90 kDa. The Vmax of the purified enzyme was 4 pmol acyldihydroxyacetone phosphate (acylDHAP) formed per minute per milligram protein and the Km(DHAP) is 70 μM when assayed at saturating concentrations of palmitoylCoA. Free coenzyme A inhibits the acyltransferase reaction with an inhibition constant (Ki) of approximately 0.76 mM. To date, this is the most highly purified DHAP acyltransferase(>3200-fold) of mammalian origin.

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.

48] Acyl/alkyldihydroxyacetone phosphate reductase from guinea pig liver peroxisomes

Publisher Summary Acylglycerone-phosphate reductase—a membrane-bound enzyme present in animal tissues—catalyzes the reduction of long-chain acyldihydroxyacetone phosphate (acyI-DHAP) or long-chain alkyl-DHAP by NADPH to the corresponding sn -glycerol 3-phosphate derivatives. In liver and other organs, the major fraction of the enzyme is present in peroxisomes. The enzyme has been solubilized and partially purified from Ehrlich ascites cell microsomes and purified to homogeneity from guinea pig liver peroxisomes. This chapter describes a method for the preparation of the homogeneous enzyme from guinea pig liver peroxisomes. The enzyme activity is assayed by measuring the radioactive lipid product formed after incubating alkyl-DHAP with B-[4- 3 H] NADPH. The purification of alkyldihydroxyacetone phosphate is discussed in the chapter. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the purified enzyme shows a single protein band of relative mobility corresponding to 60,000 molecular weight. On gel filtration, in the presence of octylglucoside, the molecular weight of the enzyme is estimated at 75,000. The enzyme has a broad pH optimum between pH 6.5 and 7.5.

Stereochemistry of the acyl dihydroxyacetone phosphate acyl exchange reaction.

The fatty acid of acyl dihydroxyacetone phosphate can be exchanged enzymatically for another fatty acid. It has been shown that this reaction proceeds by cleavage of the oxygen bound to C-1 of the dihydroxyacetone phosphate (DHAP) moiety rather than by the more common cleavage at the acyl to oxygen bond. In the present study, the stereochemistry of this reaction was defined further; using deuterated substrates and fast atom bombardment-mass spectrometry, it was shown that the fatty acid exchange involves the stereospecific labilization of the pro-R hydrogen at C-1 of the DHAP moiety of acyl DHAP. The mechanism of ether bond formation, in which acyl DHAP is converted to O-alkyl DHAP, also proceeds via labilization of the pro-R hydrogen and cleavage of the fatty acid at the C-1 to oxygen bond. In addition, other workers have provided evidence that the enzyme responsible for the exchange reaction is O-alkyl DHAP synthetase. Therefore, the present results support the hypothesis that the acyl exchange is the reverse reaction of the first step in O-alkyl DHAP synthesis; in both of these reactions the pro-R hydrogen of C-1 of the DHAP moiety of acyl DHAP and the fatty acid moiety are labilized with cleavage of the fatty acid at the DHAP C-1 to oxygen bond.

Changes in the activities of dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferases in rat liver under various conditions

Activities of enzymes relating to the acyl dihydroxyacetone phosphate (acyl DHAP) pathway were determined in rat liver under conditions known to elevate the peroxisomal β-oxidation activity. In fasted and streptozotocin-induced diabetic rats, DHAP acyltransferase activity showed a small but significant increase, though the activities of glycerol-3-phosphate (GP) acyltransferase and alkyl DHAP synthase were not changed. After 2 weeks, feeding of 20% partially hydrogenated marine oil, the activity of DHAP acyltransferase also increased to 140% of the control. The feeding of 0.25% clofibrate and 2% di(2-ethylhexyl)phthalate (DEHP) increased the activities of both DHAP and GP acyltransferases by 2- to 3-fold, whereas alkyl DHAP synthase activity decreased under the same conditions. A fractionation study showed that the increases in the activities of DHAP acyltransferase and acyl /alkyl DHAP reductase in the liver of rats treated with DEHP occurred mainly in peroxisomes and microsomes, respectively. The phospholipid contents per mg protein of the isolated hepatic peroxisomes from rats were as follows (percent of the control): fasting, 62%; diabetic, 69%; high fat-diet, 89%; clofibrate-treated, 126%; DEHP-treated, 119%. These results suggest that glycerophospholipid metabolism might also be controlled by peroxisomal enzymes under physiological and pathological conditions.

Synthesis of regiospecifically labeled [18O]glycolic acid and [18O]acyldihydroxyacetone phosphate.

Methods are detailed for the preparation of [2-18O]glycolate from chloroacetic acid and for the direct conversion of these intermediates to regiospecifically labeled [2-18O]-2-O-acylglycolic acids containing approximately 90% 18O at the C-O-acyl bond. Methods are also detailed for optimization of reaction conditions and yields for each synthetic step in previously published methods for the preparation of 1-O-acyldihydroxyacetone-3-O-phosphate (DHAP) from acyloxyacetic acid (i.e., 2-O-acylglycolic acid), where acyl is tetradecanoyl, hexadecanoyl, or heptadecanoyl. The optimized reaction conditions generate 1-O-acyl DHAP in its acid form, both in high overall yield and in high purity, without requiring a final chromatographic purification of the product, 1-O-acyl DHAP. Combining these new methods, efficient and facile preparations of regiospecifically labeled [1-18O]-1-O-hexadecanoyl DHAP and [1-18O]-1-O-heptadecanoyl DHAP have now been demonstrated, in which approximately 90% 18O is specifically located only at the C-O-acyl position. Some mechanistic postulates are offered to account for the optimized yields, regioselectivities, and high 18O incorporation which are observed in the reactions we have employed to generate 1-O-acyl DHAP from glycolate intermediates.

O-alkyl lipid synthesis: The mechanism of the acyl dihydroxyacetone phosphate fatty acid exchange reaction

We have previously provided evidence for a mechanism by which acyl DHAP is converted enzymatically to O-alkyl DHAP. This mechanism involves, in part, the formation of an endiol of acyl DHAP, loss of the fatty acid by splitting of the DHAP carbon-1 to oxygen bond and the gain of a long chain fatty alcohol. It has been shown that acyl DHAP can exchange its fatty acid for one in the medium, presumably by the mediation of O-alkyl DHAP synthase. In the present ivestigation we have shown that the fatty acid which is gained by acyl DHAP in the exchange process retains both carboxyl oxygens, as predicted by our postulated mechanism. This reaction is exceptional because the usual action of acyl hydrolases is to cleave at the oxygen to acyl bond.

Peroxisomal localization of glucose-6-phosphate dehydrogenase and pyrophosphate-stimulated dihydroxyacetone-phosphate acyltransferase in mouse kidney.

1. The subcellular localization of dihydroxyacetone-phosphate acyltransferase (DHAPAT) (assayed in the presence of pyrophosphate) and glucose-6-phosphate dehydrogenase (NADP+-dependent) activity in mouse kidney was investigated by density-gradient centrifugation. 2. DHAPAT has a predominantly peroxisomal distribution, and the activity in purified peroxisomes is stimulated by various organic and inorganic phosphate-containing compounds. The pH optimum is acid. 3. Approx. 10% of the cellular NADP+-dependent glucose-6-phosphate dehydrogenase activity is associated with peroxisomal fractions and may provide a source of NADPH for the peroxisomal reduction of acyl-dihydroxyacetone phosphate formed by DHAPAT activity.

Alkyldihydroxyacetonephosphate synthase mechanism: 18O studies of fatty acid release from acyldihydroxyacetone phosphate.

Alkyldihydroxyacetonephosphate synthase (alkyl-DHAP synthase) catalyzes the exchange of the ester-linked fatty acid of 1-O-acyldihydroxyacetone phosphate (1-O-acyl-DHAP) for a fatty alcohol that is attached in an ether linkage to form 1-O-alkyldihydroxyacetone phosphate (1-O-alkyl-DHAP). In our continuing investigation of the mechanism of this enzyme, we have examined the fatty acid released during the reaction. In contrast to the reports of others using whole microsomes, we found that the cleavage of fatty acid by purified preparations of alkyl-DHAP synthase was dependent on the presence of the cosubstrate, fatty alcohol. Furthermore, the amount of fatty acid produced was equivalent to the alkyl-DHAP formed. Our previously proposed detailed mechanism for alkyl-DHAP synthase predicted that the fatty acid should retain both of the carboxyl ester oxygens upon cleavage. Reactions carried out with palmitoyl-[18O]DHAP as substrate yielded [18O]palmitic acid as the product in agreement with this scheme.

Pancreatic islets synthesize phospholipids de novo from glucose via acyl-dihydroxyacetone phosphate

There is considerable evidence that an increased turnover of phosphoinositides and phosphatidic acid accompanies stimulus-induced insulin release. As glucose metabolism via glycolysis produces precursors for phospholipid synthesis, the time course of incorporation of [U 14C] labelled glucose was measured to determine the pathways of triose carbon incorporation into phospholipids in the islet. Cultured islets were stimulated with glucose 2.7 or 33 mM. The labelled phospholipids present after stimulation were acyldihydroxyacetone phosphate, lysophosphatidic acid, phosphatidic acid and phosphatidylinositol. Acyl-dihydroxyacetone phosphate rose promptly within 1 minute of raising the glucose concentration and was the primary acylated triose labelled during the first 15 minutes. It was possible to show in vitro conversion of [U 14C] glucose-derived acyl-dihydroxyacetone phosphate to lysophosphatidic acid and phosphatidic acid in the presence of NADPH (100 μM), indicating the presence in the islet of acyl-dihydroxyacetone phosphate: NADP oxidoreductase and acyl CoA:1 acylglycerol-3-phosphate acyl transferase, respectively. This study suggests that de novo synthesis of phosphatidic acid provides a link between glucose metabolism and the release of insulin.

Estimation of acyldihydroxyacetone phosphate and lysophosphatidate in animal tissues

Chemical and enzymatic methods have been developed to measure small quantities (10 −8−10 −10 mol) of acyldihydroxyacetone phosphate in animal tissues. Lipids extracted from tissue samples with acidic CHCl 3/ methanol were subjected to solvent partitioning at two different pH values for partial purification of keto-lipid from other lipids. This lipid was then estimated radiometrically either by chemical reduction with NaB 3H 4 or by enzymatic reduction with [4B- 3H]NADPH using a partially purified acyldihydroxyacetonephosphate reductase (EC 1.1.1.101). Thin-layer chromatography revealed the presence of a number of 3H-labeled lipids in the NaB 3H 4-reduced product and further purification of the product was necessary to estimate the amount of acyl[2- 3H]glycerol 3-phosphate formed. The enzymatic reduction was very specific for acyl/alkyldihydroxy acetone phosphate. The amounts (nmol/g) of these keto-lipids estimated in different tissues by the enzymatic method were 10.06 ± 0.64 (guinea pig liver), 4.3 ± 0.15 (rat liver), 2.1 (rat testis), 1.5 (rad kidney) and 1.2 (rat brain). Monoacylglycerol 3-phosphate, i.e., lysophosphatidic acid, which was co-purified with acyldihydroxyacetone phosphate, was found to be present in relatively larger amounts in tissues. The amounts (nmol/g) of this lipid, estimated by enzymatically measuring the amounts of sn-glycerol 3-phosphate released after alkaline methanolysis of the partially purified lipid extracts, were 143 (guinea pig liver), 58 (rat liver), 53 (rat kidney) and 92 (rat brain). Stearic acid (18:0) was found to be the major (65%) fatty acid present in the lysophosphatidate purified from guinea pig liver.

The mechanism of ether bond formation in O-alkyl lipid synthesis in Ehrlich ascites tumor. Unusual cleavage of the fatty acid moiety of acyl dihydroxyacetone phosphate.

We have previously presented evidence for the formation of 1-O-alkyl dihydroxyacetone-P from acyl dihydroxyacetone-P via the initial formation of an intermediate 1-O-acyl endiol of acyl dihydroxyacetone-P. This reaction involves a stereospecific exchange of the pro-R hydrogen of the acyl dihydroxyacetone-P moiety without change in configuration. The fatty acid is replaced by a long chain fatty alcohol which retains the oxygen of the primary carbinol. In the absence of fatty alcohol, water substitutes and the product is dihydroxyacetone-P which has also exchanged the pro-R hydrogen with a hydrogen from the medium. An absolute requirement of the proposed mechanism is that the loss of the fatty acid must proceed via an unusual cleavage of the dihydroxyacetone-P C-1 to oxygen bond instead of the usual cleavage at the fatty acid acyl to oxygen bond. In the present investigation, we have synthesized hexadecanoyl dihydroxyacetone-P containing oxygen-18 exclusively at the dihydroxyacetone-P C-1 oxygen. Using this substrate, we have shown that cleavage of hexadecanoyl dihydroxyacetone-P at the C-1 to oxygen bond is linked to O-alkyl dihydroxyacetone-P synthesis. Inhibition of O-alkyl lipid synthesis by means of magnesium or NADPH inhibited the unusual cleavage. At the same time, we have shown that there was hydrolysis of acyl dihydroxyacetone-P which proceeded by the usual mechanism and which was not related to synthesis of O-alkyl dihydroxyacetone-P.

Inhibitors of ether lipid biosynthesis

During biosynthesis of ether lipids, fatty alcohols may add covalently to ene-diol esters that would result from isomerization of acyl-dihydroxyacetone phosphate. Palmitoylation of 1,2,3-trihydroxyeicosane 1-phosphate, obtained by epoxidation of the product obtained by vinyllithiation of octadecanal, yields stable analogs of the high-energy intermediates that would be expected to result from alcohol addition. These analogs, in which an alkyl group replaces the ether alkoxyl group of the intermediates, inhibit formation of hexadecyl-dihydroxyacetone phosphate in a microsomal system from Ehrlich ascites cells. The parent compound is without effect.

Hydrogen exchange in the formation of dihydroxyacetone phosphate from acyl dihydroxyacetone phosphate in O-alkyl lipid synthesis in Ehrlich ascites tumor cell microsomes.

We have previously presented evidence that acyl dihydroxyacetone phosphate is converted to O-alkyl dihydroxyacetone phosphate via an endiol intermediate which then accepts a fatty alcohol to form O-alkyl dihydroxyacetone phosphate. We have further proposed that, in the absence of fatty alcohol, the endiol derivative of acyl dihydroxyacetone phosphate reacts with water to form dihydroxyacetone phosphate. In support of this hypothesis, we have shown, in an O-alkyl generating system, that the amount of hydrogen released from acyl dihydroxyacetone phosphate in the formation of the endiol is greater than the amount of hydrogen lost from the total lipid present at the end of incubation. The discrepancy is greater in the absence of added hexadecanol. The balance of the hydrogen loss can be accounted for by the formation of a non-lipid substance which was identified as dihydroxyacetone phosphate.

Fatty acid specificities of microsomal acyltransferases esterifying positions-1 and -2 of acylglycerols in mammary glands from lactating rats.

The acyl specificities of several acyltransferases located in the microsomal fraction of lactating rat mammary gland have been investigated using palmitate and oleate as substrates along with CoA, ATP and Mg(2+), bovine serum albumin and NaF. With either sn-glycerol 3-phosphate or dihydroxyacetone phosphate (plus NADPH) as acyl acceptor, phosphatidic acid containing palmitate preferentially esterified at position-2 and oleate at position-1 was the major product. Dihydroxyacetone phosphate and sn-glycerol 3-phosphate competitively inhibited each other's acylations, suggesting that a single enzyme might be responsible for both esterifications and oleate was the preferred substrate for the formation of acyldihydroxyacetone phosphate. The specificities of the acyl-CoA-1-monoacyl-sn-glycerol 3-phosphate and the acyl-CoA-2-monoacyl-sn-glycerol 3-phosphate acyltransferases were also studied. The specificities observed combined with the relative velocities of these reactions suggest that phosphatidic acid is formed in the mammary gland with the first acylation occurring at position-1 favouring oleate followed by the second acylation at position-2 favouring palmitate. This is consistent with the unusual structure found in the triacylglycerols of rat milk. When a mouse liver microsomal fraction was used the opposite specificities were observed consistent with the structure of the triacylglycerols of mouse liver. The microsomal acylation of the monoacyl-sn-glycerol 3-phosphocholines was also investigated. Although no marked acyl specificity could be detected when the 2-monoacyl-sn-glycerol 3-phosphocholine was used as the acyl acceptor, both oleate and linoleate were esterified in preference to palmitate to the 1-monoacyl-sn-glycerol 3-phosphocholine.