Stereospecific synthesis and enzyme studies of CDP-diacylglycerols

The fatty acid specificity of two enzymes that metabolize CDPdiacylglycerol, CDPdiacylglycerol hydrolase (EC 3.6.1.26) and CDPdiacylglycerol: inositol phosphatidyltransferase (EC 2.7.8.11), has been examined in guinea pig brain. Mixed CDPdiacylglycerols were stereospecifically synthesized by the following sequence: (i) hydrolysis of a homodiacyl lecithin to 1-acyl lysoPC by action of snake venom phospholipase A2, (ii) reacylation with the anhydride of the desired second fatty acid and dimethylaminopyridine, (iii) hydrolysis of the resultant heterodiacyl lecithin to phosphatidate with cabbage phospholipase D, and (iv) reaction of phosphatidate with CMPmorpholidate to give CDPdiacylglycerol. CDPdiacylglycerol: inositol phosphatidyltransferase showed the following rates of conversion of 40-microM suspensions of CDPdiacylglycerol in 0.15% Triton X-100 to phosphatidylinositol relative to the 1-stearoyl-2-oleoyl derivative (100%): dipalmitoyl, 70%; distearoyl, 38%; diarachidonoyl, 9%; 1-arachidonoyl-2-stearoyl, 6%; 1-stearoyl-2-arachidonoyl, 4%. These results indicate that the composition of isolated phosphatidylinositol and related lipids is not explained by the fatty acid specificity of the biosynthetic enzymes and supports the intervention of a deacylation-reacylation sequence. The rates of hydrolysis of the synthetic CDPdiacylglycerols at 76 microM, in 0.3% Triton X-100, by the CDPdiacylglycerol hydrolase relative to the 1-stearoyl-2-oleoyl derivative (100%) were: dipalmitoyl, 70%; distearoyl, 32%; 1-arachidonoyl-2-stearoyl, 30%; 1-stearoyl-2-arachidonoyl, 28%; diarachidonoyl, 22%. Inhibition of this enzyme by AMP was shown to be non-competitive, with a Ki of 40 microM. The lysosomal localization of the mammalian hydrolase was confirmed.     

Properties of levansucrase fromBacillus circulans

A polysaccharide-producing bacterium was isolated from cane sugar. It was identified asBacillus circulans and produced levansucrase at pH and temperature optima of 5–7 and 40°C respectively. The enzyme is extracellular and inducible with sucrose. It possesses initial hydrolytic and transferase activities that can be altered by modifying reaction conditions. Levansucrase was recovered from the fermentation broth by extraction with polyethylene glycol (1500 Da). Further purification resulted in an enzyme with a molecular mass of 52 kDa and a pI of 4.7. At high sucrose concentration (300 mM), the transferase activity but not the hydrolase activity were inhibited. Levan increased the transferase activity but had no effect on the hydrolytic activity. The levansucrase had high transferase activity with maltose, galactose and lactose and moderate activity towards sorbitol and glycerol.     

Fatty acid composition of cholesterol esters in brains of patients with Schilder's disease, G M1 -gangliosidosis and Tay-Sachs disease, and its possible relationship to the -position fatty acids of lecithin

Abstract— Cholesterol esters were isolated from cerebral cortex and white matter of patients with Schilder's disease, G_M1-gangliosidosis and Tay-Sachs disease, and the fatty acid composition was determined by gas-liquid chromatography. The fatty acid composition was similar among the three pathological conditions, but it was entirely different from that reported for cholesterol esters of normal brain. Lecithin and ethanolamine phospholipids were isolated from the same brain specimens, treated with snake venom phospholipase A, and the fatty acids at the a’and β-positions of the glycerol moiety were determined separately. The fatty acid composition of cholesterol esters was similar to that of the β-position fatty acids of lecithin of white matter in all samples, and was quite different from those of the a'-position of lecithin, or of the a’or β-position of ethanolamine phospholipids. The results indicate that the source of fatty acids for cholesterol esterification in nonspecific sudanophilic demyelination is different from that in normal brain, and that the most likely source is the β-linked fatty acids of lecithin. There are two possible enzymic mechanisms; activation of phospholipase A and subsequent esterification of the liberated β-position fatty acids to cholesterol, or direct transacylation by lecithin-cholesterol acyl transferase.     

Isolation of myoinositol from solutions of enzymatic biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D on phase interface in water-organic solvent systems was studied. Optimal conditions for phosphatidylinositol synthesis in water-organic solvent heterogeneous system were determined. The rapid separation of the hydrophobic components, phospholipids, from water-soluble products, alcohols, was observed in the systems with organic solvents. Displacement of myo-inositol from phosphatidylinositol by methanol, alcohol substrate, added to the reaction medium was shown in hexane-water system. Myo-inositol was isolated from the mixture of its isomers by two-stage transferase reaction catalyzed by phospholipase D.     

Isolation of myo-Inositol from Solutions by Enzymatic Biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D was studied at interfaces in water–organic solvent systems. Optimum conditions were determined for phosphatidylinositol synthesis in heterogeneous water–organic solvent systems. Hydrophobic components (phospholipids) were readily separated from water-soluble products (alcohols) in systems with organic solvents. In the hexane–water system, addition of methanol (an alcohol substrate) to the reaction medium displaced myo-inositol from the molecule of phosphatidylinositol. myo-Inositol was isolated from the mixture of its isomers using a two-step transferase reaction catalyzed by phospholipase D.     

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.     

Influence of phospholipid environment on the phosphatidylethanolamine: ceramide-phosphorylethanolamine transferase activity in rat liver plasma membranes.

1. Plasma membranes were treated with phospholipase A2, phospholipase C or phospholipase D. The phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was deactivated by phospholipase C treatment, whereas phospholipase A2 and phospholipase D did not affect the enzyme. 2. Incorporation of phosphatidylethanolamine and phosphatidylglycerol into partially delipidated plasma membranes resulted in significant stimulation of the transferase, whereas inclusion of sphingomyelin and phosphatidylserine suppressed the enzyme activity. Our results suggest that phosphatidylserine is a regulator of sphingomyelin level in membranes. 3. The activity of phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was not influenced by the fluidity of its lipid environment.     

Modulation of phospholipase D-mediated phosphatidylglycerol formation by differentiating agents in primary mouse epidermal keratinocytes

The major component of the epidermis, keratinocytes, must continuously proliferate and differentiate to form the mechanical and water permeability barrier of the skin. Our previous data have suggested a potential role in these processes for phospholipase D (PLD), an enzyme that hydrolyzes phospholipids to generate phosphatidic acid. In the presence of primary alcohols, PLD also catalyzes a transphosphatidylation reaction to produce phosphatidylalcohols, and this characteristic has been exploited to monitor the activity of PLD in intact cells. In this report, PLD was demonstrated to utilize the physiological alcohol glycerol to form phosphatidylglycerol (PG) in vitro. In intact primary murine epidermal keratinocytes treated for 24 h with elevated extracellular Ca(2+) levels, but not 1,25-dihydroxyvitamin D(3), incubation with radioactive glycerol resulted in an increase in PLD-mediated radiolabeled PG production. This effect was dose-dependent and biphasic, with maximal PG formation detected after exposure to an intermediate (125 microM) Ca(2+) concentration. Furthermore, the biphasic nature of the response was due, in part, to a corresponding biphasic change in glycerol uptake. Finally, short-term treatment of keratinocytes with phorbol 12-myristate 13-acetate (PMA) failed to increase PG synthesis and inhibited glycerol uptake. Since (1) PMA is reported to activate PLD-1 to a greater extent than PLD-2, (2) 1,25-dihydroxyvitamin D(3) increases the expression/activity of PLD-1 in keratinocytes, and (3) PLD-2 is co-localized with a glycerol channel in keratinocyte membrane microdomains, we speculate that radiolabeled PG production from radioactive glycerol is a measure of PLD-2 activation in these cells. Our results also suggest that PLD-mediated PG synthesis may be regulated at the level of both PLD activity and alcohol substrate availability via changes in glycerol uptake.     

Effectors Differently Modulating the Dextransucrase Activity of Leuconostoc mesenteroides

Effects of various compounds on the dextransucrase (EC 2.4.1.5) from Leuconostoc mesenteroides was evaluated based on the two catalytic activities of enzyme, that is the hydrolase activity for the substrate, sucrose, and the transferase activity of a d-glucosyl group to an acceptor molecule. The effectors were grouped into six categories by their activation or inhibition of the sucrase and transferase activities of dextransucrase. Type I-A inhibited both activities, type I-B inhibited the sucrase activity, and type I-C inhibited the transferase activity. Type A-A activated both the hydrolase and transferase, and type A-B activated only the transferase. Antagonistic modulation (type IA-A), was shown by methyl α-d-glucoside and glycerol, which activated the sucrase and inhibited the transferase. A double reciprocal plot for dextran gave a biphasic pattern which led to Ki values for each limb. Based on the biphasic kinetics and the action of antagonistic effectors, the regulation of dextran synthesis was discussed.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Endocannabinoid metabolism in human glioblastomas and meningiomas compared to human non-tumour brain tissue

The endogenous levels of the two cannabinoid receptor ligands 2-arachidonoyl glycerol and anandamide, and their respective congeners, monoacyl glycerols and N-acylethanolamines, as well as the phospholipid precursors of N-acylethanolamines, were measured by gas chromatography-mass spectrometry in glioblastoma (WHO grade IV) tissue and meningioma (WHO grade I) tissue and compared with human non-tumour brain tissue. Furthermore, the metabolic turnover of N-acylethanolamines was compared by measurements of the enzymatic activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolysing phospholipase D and fatty acid amide hydrolase in the same three types of tissue. Glioblastomas were characterized by enhanced levels of N-acylethanolamines (eightfold, 128 +/- 59 pmol/micromol lipid phosphorus) including anandamide (17-fold, 4.6 +/- 3.1 pmol/micromol lipid phosphorus) and several species of N-acylphosphatidylethanolamines (three to eightfold). This was accompanied by a more than 60% reduction in the enzyme activities of N-acylphosphatidylethanolamine-hydrolysing phospholipase D and fatty acid amide hydrolase. By contrast, meningiomas were characterized by a massively enhanced level of 2-monoacyl glycerols (20-fold, 2293 +/- 361 pmol/micromol lipid phosphorus) including 2-arachidonoyl glycerol (20-fold, 1524 +/- 361 pmol/micromol lipid phosphorus). This was accompanied by an enhanced in vitro conversion of phosphatidylcholine to monoacyl glycerol (fivefold). The enhanced level of the 2-arachidonoyl glycerol, anandamide and other N-acylethanolamines detected in the two types of tumour tissue may possibly act as endogenous anti-tumour mediators by stimulation of both cannabinoid and non-cannabinoid receptor-mediated mechanisms.     

Purification of a phospholipase from Botrytis and its effects on plant tissues

A phospholipase from Botrytis cinerea, grown in pure culture, was purified more than 1000-fold. Whilst it possessed no acyl hydrolase activity toward a variety of p-nitrophenyl fatty acyl derivatives, phosphatidyl choline (lecithin) acted as a substrate; the enzyme being of either type ‘A’ or ‘B’ specificity. When the purified enzyme was applied to washed beetroot discs, betacyanin leakage was induced. Loss of substances which absorb at 260 nm also occurred when washed potato tuber discs were incubated with the enzyme. Incubation with a lysosome-enriched fraction from potato sprout tissues resulted in increased acid phosphatase activity in the incubation supernatant. The phospholipase had no macerating effect on a variety of plant tissues, nor did it cause disruption of isolated protoplasts from these tissues. Studies with mitochondria from mung beans revealed no effects of the enzyme upon the respiratory activity of these organelles. The result suggested that a major site of action of the B.cinerea phospholipase was the lysosomes.     

One step purification of peanut phospholipase D by precipitation with alginate

A simple titrimetric assay with soybean lecithin has been used for screening phospholipase D activity from some plant sources, viz. peanut, wheat germ, cabbage and carrot. The enzyme from peanut has been purified by binding to alginate which is a water soluble polymer. The purification consisted of co-precipitation of enzyme with alginate upon addition of 0.06 M Ca⁺⁺. The enzyme was eluted from the polymer using 0.2 M sodium chloride. The activity recovery was 61% with 34 fold purification.     

Neutral and polar lipid metabolism in liver microsomes of growing rats fed a calcium-deficient diet

We studied the incorporation of (14)C-labeled fatty acids and glycerol into different classes of glycerolipids in an in vitro system containing liver microsomes from growing Wistar rats fed a calcium-deficient (CaD; 0.5 g/kg) diet for a 60-day period. Desaturase activities and incorporation of the elongation-desaturation metabolites into specific neutral and polar glycerolipids were also studied and correlated with the activities of various enzymes involved in complex lipid metabolism (acyl-CoA synthase, acyl-CoA hydrolase, DAG-acyltransferase, DAG-kinase, lysophospatidate-acyl-CoA transferase, phosphatidate-phosphohydrolase and phospholipase A(2)). Low calcium condition led to a significant increase in the incorporation (relative amounts and specific activities) of both labeled fatty acids and glycerol with a preferential increase of labeling in neutral lipids rather than in phospholipids. Acyl-CoA synthetase, diacylglycerol acyltransferase and diacylglycerol-3-P acyltransferase activities were increased in low calcium microsomes while diacylglycerol kinase, phospholipase A(2) and palmitoyl-, stearoyl-, linoleyl-, alpha-linolenyl, and eicosatrienoyl-desaturases were decreased. The modifications observed in the interlipid and lipid/protein relationships, enzyme activities, and pattern of incorporation of labeled precursors into each glycerolipid class, suggest that decreased intake of calcium should be considered as a harmful risk factor for the development of cardiovascular diseases.     

In vitro synthesis of phosphatidylinositol and phosphatidylcholine by phospholipase D

Phosphatidylinositol (PI) was prepared from egg lecithin by a one-step transphosphatidylation reaction catalysed by phospholipase D in the presence of myo-inositol. Similarly phosphatidylcholine (PC) has been synthesized by the same technique from egg phosphatidylethanolamine using phospholipase D and choline chloride.The yield of PI was ca 25 % and that of PC ca 28 %. The transphosphatidylase function of phospholipase D offers a useful route for the synthesis of different classes of phospholipids.     

Phospholipid removal during degradation of rat plasma very low density lipoprotein in vitro.

The hydrolysis of glycerophospholipids in very low density lipoprotein by enzyme(s) released into circulation after the injection of heparin to rats was studied. [32P]Lysolecithin was formed rapidly from [32P]lecithin when very low density lipoprotein, labeled biosynthetically with 32P, was incubated with postheparin plasma. The [32P]lysolecithin was associated with the plasma protein fraction of density greater than 1.21 g/ml, whereas [32P]lecithin exchanged between very low and high density lipoproteins. Inhibition of the plasma lecithin: cholesterol acyl transferase activity did not change the excess [32P]lysolecithin formation in postheparin plasma, and only a negligible amount of radioactivity was associated with blood cells when the incubation was repeated in whole blood. Analysis of the results has demonstrated that phospholipids are removed from VLDL by two pathways: hydrolysis of glycerophospholipids by the heparin-releasable phospholipase activity (greater than50%) and transfer to high density lipoproteins (less than50%). The tissue origin of the postheparin phospholipase was studied in plasma obtained from intact rats and supradiaphragmatic rats using specific inhibitors of the extrahepatic lipase system (protamine sulfate and 0.5 M NaCl). The phospholipase activity could be ascribed to both the hepatic and extrahepatic lipase systems. It is concluded that hydrolysis of glycerophospholipids is the major mechanism responsible for the removal of phospholipids from very low density lipoprotein during the degradation of the lipoprotein. It is suggested that phospholipid hydrolysis occurs concomitantly with triglyceride hydrolysis, predominantly in extrahepatic tissues.     

Delayed activation of phospholipase D by gonadotropin-releasing hormone in a clonal pituitary gonadotrope cell line (alpha T3-1).

Stimulation of cultured pituitary cells from a gonadotrope lineage (alpha T3-1) by the gonadotropin-releasing hormone agonist analog [D-Trp6]GnRH (GnRH-A) resulted in a manifold increase in accumulation of phosphatidylethanol, a specific product of phospholipase D phosphatidyl transferase activity when ethanol is the phosphatidyl group acceptor. Levels of the natural lipid product of phospholipase D, phosphatidic acid, were increased 2-3-fold. Activation of phospholipase D by GnRH-A was dose- and time-dependent and was blocked by a GnRH receptor antagonist [D-pClPhe2,D-Trp3.6]GnRH. GnRH-A stimulated phospholipase D activity after a lag of 1-2 min. We conclude that in alpha T3-1 gonadotropes GnRH receptor occupancy results in delayed activation of phospholipase D which could participate in late phases of gonadotrope regulation by the neurohormone.     

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

SENSITIVE TO FREEZING 2 (SFR2) is classified as a family I glycosyl hydrolase but has recently been shown to have galactosyltransferase activity in Arabidopsis thaliana. Natural occurrences of apparent glycosyl hydrolases acting as transferases are interesting from a biocatalysis standpoint, and knowledge about the interconversion can assist in engineering SFR2 in crop plants to resist freezing. To understand how SFR2 evolved into a transferase, the relationship between its structure and function are investigated by activity assay, molecular modeling, and site-directed mutagenesis. SFR2 has no detectable hydrolase activity, although its catalytic site is highly conserved with that of family 1 glycosyl hydrolases. Three regions disparate from glycosyl hydrolases are identified as required for transferase activity as follows: a loop insertion, the C-terminal peptide, and a hydrophobic patch adjacent to the catalytic site. Rationales for the effects of these regions on the SFR2 mechanism are discussed.     

Phospholipase D activation by the mitogens platelet-derived growth factor and 12-O-tetradecanoylphorbol 13-acetate in NIH-3T3 cells

The effect of mitogens on phospholipase D activity was investigated in NIH-3T3 fibroblasts by measuring the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity when 1-propanol acts as the phosphatidyl group acceptor. Platelet-derived growth factor (PDGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated phosphatidylpropanol production by the cells. The dose-response relationships for activation of phospholipase D and stimulation of thymidine incorporation by PDGF and TPA were comparable. The possibility that activation of phospholipase D is utilized by mitogens as a trans-membrane pathway for signalling cell growth is discussed.     

Release of dialkylglycerol from purple membrane phospholipids by phospholipase D

When the major polar lipid of purple membrane, a dialkyl analogue of phosphatidyl glycerophosphate, is treated with phospholipase D under the usual assay conditions for this enzyme, the reaction yields dialkylglycerol and glycerol bisphosphate, i.e. the kind of products that would be expected from a phospholipase C reaction. The effect is seen both in native purple membranes and with the pure phospholipid in the form of liposomes. The specific activity and kinetic parameters Km and Vmax of phospholipase D for the purple membrane phospholipid are similar to those for egg phosphatidylcholine. The presence of phospholipase C impurities in the phospholipase D preparations has been ruled out as an explanation for the above observations. A hypothesis is suggested, taking into account the peculiar headgroup structure of the bacterial lipid, to explain the seemingly anomalous enzyme behavior.