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 μmol/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.     

Biosynthesis of pulmonary surfactant: Comparison of 1-palmitoyl-sn-glycero-3-phosphocholine and palmitate as precursors of dipalmitoyl-sn-glycero-3-phosphocholine in adenoma alveolar type II cells

Urethan-induced pulmonary adenomas of mice are composed of cells that appear to be morphologically identical to alveolar type II cells and synthesize disaturated diacyl-sn-glycero-3-phosphocholine, the major component of pulmonary surfactant. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid were compared as precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma type II cells by incubating both substrates with whole adenomas. When the precursors were compared at equal concentrations (100 μm) in the presence of albumin (1 mg/ml), the rates of incorporation of 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid into diacyl-sn-glycero-3-phosphocholine were 5.2 and 2.9 nmol/min · g tissue, respectively. The concentration of monoacyl-sn-glycero-3-phosphocholine (lysolecithin) in the blood plasma of BALB/c mice was 150 μm. In short-term labeling experiments, the label in disaturated diacyl-sn-glycero-3-phosphocholine was equally distributed between the sn-1 and sn-2 positions when 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine was the precursor, whereas 75 to 80% was in the sn-2 position when [1-¹⁴C]palmitic acid was the precursor. The ratios are consistent with incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine via the lysolecithin:lysolecithin transacylase reaction and incorporation of palmitate via acylation of 1-palmitoyl-sn-glycero-3-phosphocholine by acyl-CoA:lysolecithin acyltransferase. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phospho-[³H-methyl]choline was incorporated into total cellular diacyl-sn-glycero-3-phosphocholine with an isotope ratio similar to that of the precursor; the disaturated species was more enriched in ¹⁴C. These findings indicate the cells take up intact monoacyl-sn-glycero-3-phosphocholine and incorporate it into diacyl-sn-glycero-3-phosphocholine. The ability of the cells to utilize intact lysophosphoglycerides for synthesis of cellular lipids was further demonstrated by showing that ether analogs, 1-alkyl-sn-glycero-3-phosphocholine and 1-alkyl-sn-glycero-3-phosphoethanolamine, are taken up and acylated by the cells. Activities of lysolecithin:lysolecithin transacylase and acyl-CoA:lysolecithin acyltransferase were measured in subcellular fractions of the adenoma type II cells; the specific activities of the enzymes were 2.1 nmol/min · mg soluble protein and 21 nmol/min · mg microsomal protein, respectively. The total activity of the acyltransferase in the cell fractions was about four-fold higher than the activity of the transacylase. Characteristics of the two enzymes were studied and are discussed. The findings indicate that exogenous 1-palmitoyl-sn-glycero-3-phosphocholine and palmitic acid both serve as efficient precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma alveolar type II cells.     

Metabolism of hepatic haem and `green pigments'' in rats given 2-allyl-2-isopropylacetamide and ferric citrate. A new model for hepatic haem turnover

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 Mg²⁺, 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.     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

An improved method for the preparation of 1,2-isopropylidene-SN-glycerol

A simple and fast route for the preparation of 1,2-isopropylidene-sn-glycerol from D-mannitol in 45% yield is described. The value of optical rotation, [α]_D²⁰ + 15.2°, is higher than usual indicating considerable racemization for other procedures. Since 1,2-isopropylidene-sn-glycerol serves as general intermediate for the synthesis of glycerides and of phosphoglycerides these lipids contain substantial amounts of the isomer, for instance 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine may consist of up to 15% of 2,3-dipalmitoyl-sn-glycerol-1-phosphocholine in earlier preparations.     

Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.     

Glycerolipid biosynthesis in rat adipose tissue. Effect of polyamines on triglyceride synthesis.

Triacylglycerol formation from sn-glycerol 3-phosphate and 1,2-diacyl-sn-glycerol was markedly elevated in the presence of spermine and spermidine. This was attributed to the activation of microsomal sn-glycerol 3-phosphate acyltransferase and 1,2-diacyl-sn-glycerol acyltransferase and to the inhibition of palmitoyl-CoA hydrolase. Spermine was more effective than spermidine, and putrescine did not stimulate triacylglycerol formation. The stimulatory effect of spermine on triacylglycerol-forming enzymes was observed in the presence of Mg²⁺ and was apparent in the presence or absence of bovine serum albumin. The activation of 1,2-diacyl-sn-glycerol acyltransferase by spermine was specific, and other diacylglycerol-utilizing enzymes were not affected under these conditions. These studies demonstrate that polyamines may be important regulators of triacylglycerol formation in adipose tissue.     

Phospholipid dependence of rat liver microsomal acyl: CoA synthetase and acyl-CoA : 1-Acyl-sn-glycero-3-phosphocholine O-acyltransferase

Summary Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used—palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA : lacyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes—1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A₂ which both participate in the deacylation-reacylation cycle.     

Some properties of diacylglycerol acyltransferase in a particulate fraction from maturing safflower seeds

The activity of diacylglycerol acyltransferase of a subcellular particulate fraction from maturing safflower seeds was remarkably stimulated by the addition of 1, 2-diacylglycerols which were previously emulsified in a gelatin solution by sonication. Metal ions were inhibitory to the reaction. Deoxycholate and diisopropyl fluorophosphate were the most effective inhibitors. Sulfhydryl groups seemed to be of limited significance in the enzyme. Both 1, 2-dioleoyl-sn-glycerol and 2, 3-dioleoyl-sn-glycerol were good substrates of diacylglycerol acyltransferase, but the 1, 3-isomer did not serve as an acyl acceptor. The enzyme showed broad specificity for synthetic rac-1, 2-diacylglycerols containing various fatty acids. However, rac-1, 2-diacetylglycerol and rac-1, 2-dibutyrylglycerol, which are soluble in water, were ineffective. The enzyme exhibited no significant specificity for saturated and unsaturated fatty acyl-CoA thioesters as acyl donors. This suggests that the fatty acid composition at the 3-position of the glycerol molecule of safflower triacylglycerols may depend on the composition of the endogenous acyl-CoA pool.     

Lysophospholipase and lysophosphatidylcholine:Lysophosphatidylcholine transacylase from rat lung: Evidence for a single enzyme and some aspects of its specificity

An enzyme preparation was isolated from rat lung cytosol with the capability to transfer the fatty acyl chain from 1-acyl-sn-glycero-3-phosphocholine to water and to another molecule of 1-acyl-sn-glycero-3-phosphocholine. The evidence presented to indicate that a single protein confers both activities includes: (a) both normal and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis showed a single protein band, and (b) heat treatment and preincubation with increasing amounts of diisopropylfluorophosphate resulted in concomitant loss of fatty acid and phosphatidylcholine formation. The enzyme converted 1-[9,10-³H₂]stearoyl-sn-glycero-3-phospho[¹⁴C-methyl]choline into phosphatidylcholine with an isotopic ³H/¹⁴C ratio twice that of the substrate, even when an excess of unlabeled fatty acid was present. The acyl group from palmitoyl-propanediol (1,3)-phosphocholine and palmitoyl-propanediol (1,3)-phosphoethanolamine could be transferred to lysophosphatidylcholine acceptor to yield phosphatidylcholine. Neither acylglycerols and cholesterol nor glycero-3-phosphate and glycero-3-phosphocholine served as acyl acceptors. Lysophosphatidylethanolamine and lysophosphatidyglycerol were converted also into the corresponding diacylphospholipids. Palmitoyllysophosphatidylcholine is preferentially converted into phosphatidylcholine when compared with stearoyllysophosphatidylcholine. The possible involvement of the enzyme in the synthesis of dipalmitoylphosphatidylcholine for the production of lung surfactant is discussed.     

Maintenance of respiratory control by beef heart mitochondria incubated at 25 degrees C: response to protective agents and to protective agents and to prior stress.

Lysophospholipase D (EC 3.1.4.-) activity was demonstrated in rat kidneys, intestines, lungs, testes, and liver. The liver enzyme was studied in greatest detail and its labeled products were identified by chemical and Chromatographic techniques. This enzyme hydrolyzes 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphoethanolamine and 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphocholine to yield 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate; the initial product is subsequently dephosphorylated by a phosphohydrolase in microsomes to form 1-[1-¹⁴C]hexadecyl-sn-glycerol. The possibility that phospholipase C and a phosphotransferase were responsible for the formation of 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate was ruled out. Neither 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine nor 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphocholine was hydrolyzed. The enzyme requires Mg²⁺, is inhibited by Ca²⁺, and is stimulated by high salt concentrations; it is localized in the microsomal fraction and has a pH optimum between 7.0 and 7.6. Inhibition by sulfhydryl reagents and protection by glutathione and dithiothreitol suggest that a sulfhydryl group is required for activity. The enzyme is inhibited by detergents and by organic solvent extraction. It appears to be tightly bound to the microsomes, since repeated freeze-thawing or sonication did not release the activity, and trypsin digestion (either in the presence or in the absence of 0.04% deoxycholate) did not destroy the activity. Lysophospholipase D was previously known to occur only in brain (R. L. Wykle and J. M. Schremmer, 1974, J. Biol. Chem., 249, 1742–1746).     

Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC)

Summary An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).     

Galactolipid synthesis in chromoplasts in vitro

Isolated chromoplasts from Narcissus pseudonarcissus flowers contain: a fatty acid synthesizing system; acyl-CoA synthetase (EC 6.2.1.3); glycero-phosphate acyltransferase (EC 2.3.1.15); acylglycero-phosphate acyltransferase; phosphatidate phosphatase (EC 3.1.3.4); diacylglycerol galactosyltransferase (EC 2.4.1.46); and diacylgalactosylglycerol galactosyltransferase, i.e. all enzymatic activities necessary for the synthesis of diacylgalactosylglycerol and diacylgalabiosylglycerol from acetate, HCO _- ³ , sn-glycerol 3-phosphate, and UDP-d-galactose. Diacylgalactosylglycerol and diacylgalabiosylglycerol, however, are synthesized from these precursors to only a very low extent in an in vitro system. This is attributed to a specificity of diacylglycerol galactosyltransferase for highly unsaturated diacylglycerols. Specificities of acyltransferase reactions were also found.     

Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br.

Triacylglycerols of both Tropaeolum majus L. and Limnanthes douglasii R. Br. are predominantly esterified with very long-chain acyl groups at each position of the glycerol backbone. In order to elucidate whether these acyl groups are directly chanelled into the triacylglycerols via the stepwise acylation of glycerol-3-phosphate, seed oil formation has been investigated in developing embryos of both plant species. [1-¹⁴C]Acetate labelling experiments using embryos at different stages of development, as well as the determination of the properties of the microsomal acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), revealed differences between the two plant species, especially with respect to the incorporation of very longchain acyl groups into the C2 position of the triacylglycerols. In microsomal fractions of developing embryos of L. douglasii both a glycerol-3-phosphate and a 1-acylglycerol-3-phosphate acyltransferase were detected which utilize very long-chain acyl-CoA thioesters as substrates. Thus, in seeds of L. douglasii very long-chain acyl groups can enter not only the C1, but also the C2 position of the triacylglycerols in the course of de-novo biosynthesis. A comparison of the properties of the acyltransferases of developing embryos with those of the corresponding activities of leaves indicates an embryo specific expression of an erucoyl-CoA-dependent microsomal 1-acylglycerol-3-phosphate acyltransferase in L. douglasii. The microsomal glycerol-3-phosphate acyltransferase of developing embryos of T. majus displayed properties very similar to those of the corresponding activity of L. douglasii. On the other hand, the microsomal 1-acylglycerol-3-phosphate acyltransferases of the two plant species showed strikingly different substrate specificities. Irrespective of the acyl groups of 1-acylglycerol-3-phosphate and regardless of whether acyl-CoA thioesters were offered separately or in mixtures, the enzyme of T. majus, in contrast to that of L. douglasii, was inactive with erucoyl-CoA. These results of the enzyme studies correspond well with those of the [1-¹⁴C]acetate labelling experiments and thus indicate that T. majus has developed mechanisms different from those of L. douglasii for the incorporation of erucic acid into the C2 position of its triacylglycerols.Abbreviations GPAT acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) - LPAT acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51) This work was supported by the Bundesministerium für Forschung und Technologie (Förderkennzeichen 0316600A).     

Leukocytic myeloperoxidase-mediated formation of bromohydrins and lysophospholipids from unsaturated phosphatidylcholines

Using MALDI-TOF mass spectrometry, we have shown that leukocytic myeloperoxidase (MPO) in the presence of its substrates (H₂O₂ and Br^?) does not induce any changes in saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Incubation of liposomes prepared from mono-unsaturated phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) with the (MPO + H₂O₂ + Br^?) system resulted in formation of bromohydrins as the main products. 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysophosphatidylcholine) was the main product of the reaction of polyunsaturated phosphatidylcholine (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) with the (MPO + H₂O₂ + Br^?) system. The formation of lysophospholipids as well as of bromohydrins was not observed when the enzyme or one of its substrates (H₂O₂ or Br^?) was absent from the incubation medium, or if an inhibitor of MPO (sodium azide) or hypobromite scavengers (taurine or methionine) were added. Thus, it can be postulated that the formation of bromohydrins as well as lysophospholipids by the (MPO + H₂O₂ + Br^?) system results from reactions of hypobromite formed during MPO catalysis with double bonds of acyl chains of phosphatidylcholine. Such destructive processes may take place in vivo in membrane-or lipoprotein-associated unsaturated lipids in centers of inflammation.     

Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis

This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich ascites cells. The highest acetylhydrolase activity for this neutral lipid was associated with the membrane fractions from Ehrlich ascites cells (> 90% of total activity); only a minimal level of activity (< 10%) was observed in the cytosol which contrasts with the cytosolic site of PAF acetylhydrolase in normal cells. Hydrolysis of 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction at pH 7.5 and 37°C gave apparent values for K_m and V_max of 45 μM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca⁺², Mg⁺² or EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral lipid acetylhydrolase does not appear to be the same enzyme that hydrolyzes acetate from platelet-activating factor. In contrast to inhibition of diacylglycerol lipase by p-chloromercuribenzoate and N-ethylmaleimide, we found no significant inhibition of acetate hydrolysis from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by either of these compounds. Also, p-nitrophenyl acetate (a nonspecific esterase substrate) failed to inhibit acetate hydrolysis of 1-[³H])hexadecyl-2-acetyl-sn-glycerol. Our studies of this enzyme would indicate that it may play an important role in regulating the levels of platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.     

Hepatic lipid metabolism: effect of spermine, albumin, and Z protein on microsomal lipid formation.

Effects of spermine, bovine serum albumin, and Z protein on microsomal lipid formation from sn-glycerol 3-phosphate and [¹⁴C]palmitoyl CoA were investigated. In the presence of these agents, microsomal lipid formation was stimulated. This was attributed to the activation of sn-glycerol 3-phosphate acyltransferase and to the inhibition of palmitoyl CoA hydrolase. In addition to palmitoyl CoA, spermine also reacted with microsomal membranes in causing their aggregation, and ATP reversed the effect of spermine. Further studies indicated that the interaction of spermine with palmitoyl CoA, rather than with microsomal membranes, was responsible for the activation of glycerolipid formation or to the inhibition of palmitoyl CoA reductase. Examination of the intravesicular distribution of sn-glycerol 3-phosphate acyltransferase and palmitoyl CoA hydrolase and the effects of structural integrity of microsomal vesicles on these two membrane-bound enzymes indicated that the activation of glycerolipid formation and the inhibition of palmitoyl CoA hydrolase by spermine, bovine serum albumin, or Z protein may be closely linked with the structural integrity of microsomal vesicles.     

Stearyl-CoA desaturase of bovine mammary microsomes

Stearyl-CoA desaturase from the microsomal fraction of lactating bovine mammary tissue had a specific activity of 0.4 nmoles oleate formed min^?1 mg^?1 protein. NADH was required for desaturase activity. However, oxidized NAD⁺ and NADP⁺ supported measurable desaturase activity. K_m values for stearyl-CoA and NADH were 25.0 μm and 3.0 μm, respectively. Desaturase was depressed by increasing concentrations of other acyl-CoA esters, i.e., palmityl-CoA and oleyl-CoA (>10 μm). Sn-1,2 diglycerides (1–2.0 μm) depressed desaturase slightly in the order 0–20%, as did l-α-glycerolphosphate (0.2–3.6 μm). 1-Acyl-sn-glycerol-3-phosphorylcholine (>0.1 μm) depressed desaturase activity markedly. Sonication of the microsomal preparation stimulated desaturase activity. The addition of ethanol depressed desaturation, and EDTA inhibited desaturation. Palmityl CoA was equally desaturated by the microsomes. The acyl-CoA desaturase was very stable when stored at ?30 °C as a freeze-dried microsomal preparation, i.e., activity was retained after 12-month storage.Labeled stearate and oleate were isolated as esters (triglycerides and phospholipids) and as free fatty acids, indicating the presence of acyl transferases and acyl-CoA hydrolase in mammary microsomes.     

Formation of optically active ether lipids from race-mic-1-O-tetradecylglycerol in plant cell cultures

Photomixotrophic rape cells in culture specifically incorporate 1-O-tetradecyl-sn-glycerol from a racemic mixture into complex alkyl glycerolipids. Thus, both neutral and ionic 1-O- alkyl-2-O-acyl-sn-glycerolipids with defined alkyl moieties can be prepared from racemic mixtures of alkylglycerols.     

On the phospholipid metabolism of glial cell primary cultures

Primary cultures prepared from newborn rat brain, consisted after 16 or 17 days mainly of astrocytes and of oligodendrocytes. 1-Alkenyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) was used as substrate for studies on the metabolism of ethanolamine-glycerophospholipids. After 3 hr incubation two main products were observed: a) 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (=ethanolamine plasmalogen) and b) 1-alkenyl-2-acyl-sn-glycero-3-phosphocholine (=choline plasmalogen). The acylation rate reached saturation at about 10 nmol substrate/mg cell protein with aV _max of 30 nmol×mg cell protein^–1×3 hr^–1. This acylated compound amounted to almost 60% of all radioactivity internalized, whereas the second product, choline plasmalogen, came to 20%. Unchanged substrate was found within the cells only in small amounts, even at maximum substrate internalization. These results were discussed in comparison with those obtained with 1-alkyl-sn-glycero-3-phosphoethanolamine under the same conditions (25).