Properties of diacylglycerol acyltransferase from spinach leaves

Diacylglycerol-acyltransferase (EC 2.3.1.20) was partially purified and characterized from spinach leaves. The enzyme had a pH optimum of 8.0 and activity was stimulated 2-fold by the addition of 20 mM Mn²⁺ or Mg²⁺. Diolein and dipalmitin were examined assn-1,2-diacylglycerol substrates. Only diolein gave detectable triacylglycerol synthesis. In addition, the saturation kinetics of the enzyme with 16∶0-CoA, 18∶0-CoA and 18∶1-CoA were examined. The highest apparent, Km was observed with 18∶1-CoA, the lowest with 16∶0-CoA. Endogenous spinach leaf glycerolipids were extracted and analyzed. The leaves contained 70 nmol triacylglycerol (g fresh weight)⁻.     

Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape

Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25mM, MgSO₄ and MgCl₂ stimulated microsomal DGAT 25- and 10-fold, respectively. AIP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg²⁺ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.     

Relation between diacylglycerol acyltransferase activity and oil concentration in soybean

Diacylglycerol acyltransferase (EC 2.3.1.20; DGAT) catalyzes synthesis of triacylglycerol from acyl-CoA and diacylglycerol. Activity of this enzyme and developmental changes in oil accumulation were estimated at various stages of seed growth in soybean germplasm with phenotypic differences in oil content. Oil deposition in seed of these genotypes followed a sigmoid pattern that was modeled to predict incremental rates of oil accumulation during seed development. A strong positive correlation was found between the estimated peak rate of oil deposition (near the mid-term of seed development) and oil concentration in mature seed. At saturating substrate levels, DGAT activity measured near the peak rate of oil deposition also was correlated positively with oil phenotype. In the latter stages of seed development, a positive correlation between estimates of enzyme activity at or below the apparent K _m for diolein and comparable oil accumulation rates was attributed to reduced synthesis of substrates and/or potential change in affinity for substrate as suggested by an increase in apparent K _m for diolein in older seed. These data indicated that DGAT activity may be a rate-limiting step in triacylglycerol synthesis. However, it is difficult to accept the idea of a single rate-limiting step at the end of a complex metabolic pathway. Because oil is a quantitatively inherited trait, several genes determine genotypic differences in oil content among soybeans. Hence, DGAT activity may be an indicator of coordinated genetic expression of gene-products in the entire glycerolipid synthetic pathway for a given genotype. In any case, results of this investigation demonstrated that genotypic differences in DGAT activity contributed to expression of genetic variation in oil content among soybean gemplasm.     

Reversible ATP-dependent inactivation of adipose diacylglycerol acyltransferase

Diacylglycerol acyltransferase from rat adipose tissue is shown to be inactivated by 30 to 40% upon incubation with adenosine 5′-triphosphate (ATP) and Mg²⁺. The activity responsible for this inactivation is associated with the cytosolic fraction, specific for ATP, prevented when ATP is substituted by β,γ-methylene-ATP, and partially blocked by 1 mM ethylenediaminetetraacetate or 40 mM NaF, but not by inhibitors of adenosine 3′,5′-cyclic-monophosphate (cAMP)-dependent protein kinase and/or protein kinase C (PKC). The cytosolic activity cannot be mimicked by (cAMP)-protein kinase nor by PKC. Inactivated diacylglycerol acyltransferase from ATP/cytosol-treated microsomes can be reactivated by incubation with partially purified protein phosphate from rat liver, and can be inactivated again by further addition of ATP in the presence of cytosol. The results suggest the existence in adipose tissue of a protein kinase other than cAMP-protein kinase or PKC, which may be involved in the regulation of triacylglycerol synthesis.     

A protein tyrosine kinase associated with the ATP-dependent inactivation of adipose diacylglycerol acyltransferase

The activity that has been previously reported to reversibly inactivate adipose glycerolphosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT)in vitro in the presence of ATP is shown here to be partially purified from adipose tissue with an apparent molecular weight of 68 kDa. The activity responsible for inactivating DGAT is associated with a kinase activity as determined by phosphate incorporation both into microsomal proteins and into a synthetic tyrosine-containing peptide as substrate for protein tyrosine kinase. Two microsomal polypeptides of 53 and 69 kDa are major substrates of this kinase. Both DGAT inactivating and kinase activities assayed from the purified sample have been found to be insensitive to the Ser/Thr kinase inhibitor H-7 while being sensitive to genistein and tyrphostin-25. A crude protein phosphatase preparation from liver was capable of reversing the effects of both activities. The purified sample was also shown to inactivate GPAT in the presence of ATP. These results suggest that a protein tyrosine kinase, in concert with a protein tyrosine phosphatase, may regulate the activities of DGAT and GPAT by a phosphorylation-dephosphorylation mechanism.     

Adipose monoacylglycerol: Acyl-coenzyme a acyltransferase activity in the white-throated sparrow (Zonotrichia albicollis): Characterization and function in a migratory bird

Although migrating birds use stored triacylglycerol as their primary fuel for flight, they must retain sufficient stores of ω6 and ω3 fatty acids to sustain reproduction after the spring migration. Hepatic monoacylglycerol:acyl-coenzyme A acyltransferase (EC 2.3.1.22) (MGAT) activity is associated with physiological periods in which lipolysis and β-oxidation are prominent, and it may also play a role in the selective retention of certain essential fatty acids. Therefore, we characterized MGAT activity in adipose tissue from the whitethroated sparrow (Zonotrichia albicollis), a migratory bird. MGAT specific activity from adipose tissue and liver, respectively, was 22.2±7.27 and 0.79±0.35 nmol/min/mg of total particulate protein. Activity did not vary seasonally or between male and female birds. Specific activity increased 4.3-fold in the presence of 75 μg of phosphatidylcholine and phosphatidylserine (1∶1, w/w). MGAT acylatedsn-1(3)-monooleoyglycerol,sn-2-monoolyelglycerol ether andsn-1(3)-monooleylglycerol ether at 7.5, 5.7 and 1.7%, respectively, of the rate observed withsn-2-monooleoylglycerol. An initial lag phase observed at low concentrations of palmitoyl-CoA was corrected by adding 2 mM MgCl₂ Mg(NO₂)₂ or CaCl₂, suggesting a requirement for divalent cations. MGAT acylatedsn-2-monolinolenoylglycerol andsn-2-monolinoleoylglycerol in preference tosn-2-monooleoylglycerol. Specificity of MGAT forsn-2-monoacylglycerols and the probable enhanced affinity forsn-2-monoacylglycerols of specific acyl chains may allow selected ω6 and ω3 fatty acids to be retained within the adipocyte, white nonessential fatty acids are released for β-oxidation in flight muscles.     

Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.     

Alteration in membrane permeability by diacylglycerol and phosphatidylcholine containing arachidonic acid

The phospholipid metabolites, stearoylarachidonylglycerol and diarachidonylglycerol, stimulate transepithelial sodium transport in frog skin epithelium. The increase in Na transport is due to an increase in the unidirectional influx of sodium, is amiloride sensitive and is prevented with pretreatment with indomethacin, mefanamic acid and phospholipase inhibitor, mepacrine. The data suggest a possible role of phospholipid metabolism and prostaglandin biosynthesis in the regulation of transepithelial ion transport.     

Decreased lecithin: Cholesterol acyltransferase activity in the plasma of hypercholesterolemic pigs

Lecithincholesterol acyltransferase (LCAT) activity levels were determined, as function of plasma total cholesterol (TC) in 13 normocholesterolemic (TC<85 mg/dL) and in 28 hypercholesterolemic (TC>98 mg/dL) pigs. The normocholesterolemic group consisted of pigs that carried apo-B allelic genes other thanLpb ⁵ and orLpb ⁸. The hypercholesterolemic group consisted ofLpb ^5/x andLpb ^5/8 heterozygous andLpb ^5/5 homozygous animals. The data reported in this study show that the LCAT activity in the plasma of hypercholesterolemic (HC) pigs (79±43 units) was significantly lower (p<0.0005) compared to the normocholesterolemic controls (175±45 units). Furthermore, LCAT activity was positively correlated with TC in the normocholesterolemic group (r=+0.54; p<0.05), whereas it was negatively correlated with TC in the hypercholesterolemic group (r=−0.73; p<0.001). Additional data obtained from incubation experiments suggest that the lower LCAT activity in hypercholesterolemic pigs may be due, at least in part, to inhibition of LCAT activity by components found in the lipoprotein-deficient fractions of the plasma of hypercholesterolemic pigs.     

Increased lysolecithin acyltransferase activity in the plasma of type II hyperlipoproteinenic patients

Lecithin-cholesterol acyltransferase (LCAT) in human plasma has been shown to acylate lysolecethin to lecithin in presence of low density lipoprotein (LDL). To determine the physological importance of LDL in activating lysolecithin acyltransferase (LAT) activity, we assayed the LAT activity in 18 hypercholesterolemic (Type II) patients and 15 control subjects. The enzyme activity was about 60% higher in the patients compared with the controls. On the other hand, the LCAT activity, measured by 2 different procedures, as well as enzyme mass, determined by radioimmuno assay, were comparable in the controls and hypercholesterolemics. The LAT activity was highly correlated with LDL levels in the plasma, but the LCAT activity and the enzyme mass had no correlation with the LDL levels. These results show that the plasma LDL is the rate-limiting activator of the enzyme, and pathological conditions, resulting in higher LDL levels, also cause higher LAT activities.     

Albumin stimulates lysophosphatidic acid acyltransferase activity in T-lymphocyte membranes

Phosphatidic acid (PtdOH) and lysophosphatidic acid (lysoPtdOH) have been shown to enhance T-lymphocyte function. However, the FA preference and influence of acyl-CoA binding proteins on lysoPtdOH and PtdOH biosynthesis are not known. Therefore, we determined glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LAT) activity in rat T-lymphocyte and liver membrane preparations in the presence of palmitoyl-CoA and oleoyl-CoA with or without BSA. We found two different properties of GPAT and LAT in whole T-lymphocyte membrane preparations relative to liver. First, T-lymphocyte basal GPAT and LAT activities were similar, whereas in liver membranes LAT activity was 10-fold higher than GPAT. Second, T-lymphocyte LAT, but not GPAT, activity was inducible (fivefold) by the addition of albumin in the presence of palmitoyl-CoA but not oleoyl-CoA. In contrast, albumin stimulated GPAT, but not LAT, activity in liver membranes in the presence of palmitoyl-CoA. These results show, for the first time, that T-lymphocyte LAT activity can be increased by the presence of an acyl-CoA binding protein, which may indicate a new important control mechanism for regulating intracellular lysoPtdOH and PtdOH levels in T-lymphocytes.     

Inhibition of diacylglycerol: CDPcholine cholinephosphotransferase activity by dimethylaminoethylp-clorophenoxyacetate

Cholinephosphotransferase [EC 2.7.8.2] activity of rat liver microsomes, with 1,2-di-0-[³H]acyl glycerol or 1-0-hexadecanoyl [U-¹⁴C]ethanediol as substrate, was inhibited byN,N-dimethylaminoethylp-chlorophenoxyacetate (centrophenoxine). Inhibition progressed in a linear fashion with increasing drug levels and was complete at 30 mM concentration. It appears that the microsomal enzyme was largely affected by the drug itself because the hydrolysis products of centrophenoxine, viz.,N,N-dimethylaminoethanol andp-chlorophenoxyacetic acid, were less inhibitory.     

Characterization of the high-melting glyceride fraction isolated from cottonseed oil stearin by fractional crystallization in isopropanol

Cottonseed oil stearin is generally produced commercially from cottonseed oil by winterization. In Egypt, the byproduct stearin is often used for lower-priced industrial applications. It can be turned into a high-value, cost-effective edible product by further fractionation to produce low-melting cottonseed oil stearin (LMCS) and a higher melting cottonseed oil stearin (HMCS). In the present investigation, isopropyl alcohol (IPA) helps to obtain good fractionation of HMCS having useful solidification and melting properties. The advantage of using IPA is that fractionation can be carried out at higher temperatures than hexane with better separation efficiency. A direct relationship between yield, quality of HMCS, and crystallization temperature during fractionation with IPA was achieved. HMCS could be fractionated from IPA at 4°C in a much higher yield but the melting point and the solid fat contents were much lower than that fractionated at 18°C. The thermal profile of the HMCS fractionated with hexane lies between those fractionated at 4 and 18°C with IPA. On the other hand, all the LMCS fractions could be considered as good quality salad oils.     

Intestinal apolipoprotein B secretion is inhibited by the flavonoid quercetin: Potential role of microsomal triglyceride transfer protein and diacylglycerol acyltransferase

Recent studies have yielded evidence that plant flavonoids reduce hepatic lipid and apolipoprotein B (apoB) secretion. However, the possible role of flavonoids in regulating lipid and apoB secretion by the intestine has not been studied. The purpose of our study was to examine the effects of quercetin, a common dietary flavonoid, on TAG and apoB secretion in a human intestinal cell-line, CaCo-2. Differentiated postconfluent CaCo-2 cells grown on filters and pretreated with quercetin for 8 h were shown by ELISA to inhibit basolateral apoB secretion in a dose-dependent manner. At 15 μM, the secretion of both apoB-100 and apoB-48 were inhibited similarly. This effect was shown to be specific, as quercetin did not affect the incorporation of [³⁵S]methionine/cysteine into secreted TCA-precipitable proteins. To determine the mechanism underlying this inhibitory effect, we examined two regulatory points: IAG availability and lipid transfer to the lipoprotein particle. Quercetin inhibited TAG synthesis under both basal and lipid-rich conditions, indicating that lipid availability is a determining factor in the regulation of apoB secretion by quercetin. The reduction was due at least in part to a decrease in diacylglycerol acyltransferase activity. We next examined lipid transfer or lipidation of the lipoprotein particle by analyzing microsomal IAG transfer protein (MTP) activity. Quercetin decreased MTP activity moderately. In summary, the data demonstrated that pharmacological concentrations of quercetin are a potent inhibitor of intestinal apoB secretion and that reduced lipid availability and lipidation in the lipoprotein assembly step are the mechanism for the suppression of apoB-containing lipoprotein secretion by quercetin in CaCo-2 cells.     

How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli

Several studies have demonstrated that it is viable to use microcrystalline preparations of water-soluble proteins as samples in solid-state NMR experiments [1-5]. Here, we investigate whether this approach holds any potential for studying water-insoluble systems, namely membrane proteins. For this case study, we have prepared proteoliposomes and small crystals of the alpha-helical membrane-protein diacylglycerol kinase (DGK). Preparations were characterised by 13C- and 15N-cross-polarization magic-angle spinning (CPMAS) NMR. It was found that crystalline samples produce better-resolved spectra than proteoliposomes. This makes them more suitable for structural NMR experiments. However, reconstitution is the method of choice for biophysical studies by solid-state NMR. In addition, we discuss the identification of lipids bound to membrane-protein crystals by 31P-MAS NMR.     

Biotransformation of glycidol by the unspecific wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase of Acinetobacter baylyi ADP1

Glycidol was biologically derivatized by the unspecific wax ester synthase/acyl coenzyme A (acyl‐CoA): diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter baylyi ADP1 into glycidyl acyl ester. Catalysis of in vitro conversion of glycidol to glycidyl acyl ester by the WS/DGAT from A. baylyi was verified by (i) a radiometric assay, (ii) thin‐layer chromatography and (iii) also by ESI‐MS. A specific activity of 50 nmol·mg^–1·min^–1 was obtained when 10 mM glycidol and 5 µM palmitoyl‐CoA were used. In vivo synthesized glycidyl acyl esters in recombinant E. coli were detected and quantified by staining with the epoxide‐specific reagent 4‐(4‐nitrobenzyl)‐pyridine. Of glycidyl acyl esters, 1.5 mg/L was obtained from the culture in the presence of 10 mM glycidol and 10 mM oleate.     

Enhancement of liver microsomal acyl CoA-lysophospholipid acyltransferase activity in pyridoxine-deficient rats

In rats deficient in pyridoxine and essential fatty acids, liver phospholipids contained less arachidonic acid and more oleic and eicosatrienoic acids than those from animals only deficient in essential fatty acids. This pattern persisted after the animals were supplemented with linoleate for 6 days. Liver oleyl and arachidonyl CoA-lysophospholipid acyltransferase activities were significantly higher in pyridoxine-deficient animals. Supplementation with linoleate for 6 days resulted in a marked increase in arachidonyl CoA-acyltransferase activity in pyridoxine-deficient rats but a relatively small increase in the supplemented animals. Differences in fatty acid composition between pyridoxine-deficient and supplemented rats can not be ascribed to lower liver transacylase activity in the deficient animals.