Fatty acid component of vitamin A ester in sheep liver

Vitamin A, when extracted along with other lipids from sheep liver, had an E1cm.1% value of 14.4, which was raised to 45.57 on removal of the phospholipids by cold acetone. Selective hydrolysis of triglycerides by an extract of acetone-dried sheep pancreas in the presence of HgCl2 as inhibitor of vitamin A esterase, followed by chromatography through alumina gave a product with E1cm.1% value of 276. This on chromatography through magnesium oxide raised the E1cm.1, value to 601.5, representing 64% pure vitamin A ester calculated as palmitate, and the total recovery was 23% of the starting oil. The purified ester preparation, when subjected to reverse-phase chromatography on silicone-impregnated paper, gave a single ultraviolet fluorescent band. The fluorescent band on hydrolysis gave only one fatty acid. This was conclusively identified to be palmitic acid.

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using H-3]inositol. P-32]orthophosphate, 3H]palmitate and C-14]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids. (C) 2010 Elsevier B.V. All rights reserved.

H2O2 has a role in cellular regulation

H2O2, in addition to producing highly reactive molecules through hydroxyl radicals or peroxidase action, can exert a number of direct effects on cells, organelles and enzymes. The stimulations include glucose transport, glucose incorporation into glycogen, HMP shunt pathway, lipid synthesis, release of calcium from mitochondria and of arachidonate from phospholipids, poly ADP ribosylation, and insulin receptor tyrosine kinase and pyruvate dehydrogenase activities. The inactivations include glycolysis, lipolysis, reacylation of lysophospholipids, ATP synthesis, superoxide dismutase and protein kinase C. Damages to DNA and proteoglycan and general cytotoxicity possibly through oxygen radicals were also observed. A whole new range of effects will be opened by the finding that H2O2 can act as a signal transducer in oxidative stress by oxidizing a dithiol protein to disulphide form which then activates transcription of the stress inducible genes. Many of these direct effects seem to be obtained by dithiol-disulphide modification of proteins and their active sites, as part of adaptive responses in oxidative stress.

Effect of thiocarbamate, urea, and uracil herbicides on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of thiocarbamates (S-ethyldipropylthiocarbamate and diallate), substituted ureas (monuron and diuron), and uracils (bromacil and terbacil) on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. The uptake of [1-14C]acetate by leaf disks was inhibited by the thiocarbamates and marginally by the substituted ureas, but not by the uracil herbicides. The uptake of [methyl-14C]choline was inhibited to a lesser extent by thiocarbamates, while the other herbicides showed a slight stimulation. The thiocarbamates almost completely inhibited uptake of [32P]orthophosphate at 1.0 mM concentration, while diuron and terbacil showed significant inhibition. [1-14C]Acetate incorporation into lipids was inhibited only by diallate. [methyl-14C]Choline incorporation into the choline phosphoglycerides was inhibited by diallate, diuron, and bromacil. The incorporation of [32P]orthophosphate into phospholipids was substantially inhibited (over 90% at 1.0 mM) by the thiocarbamates, but not by the other herbicides. [35S]Sulfate incorporation into sulfoquinovosyl diglycerides was markedly inhibited only by the thiocarbamates. Fatty acid synthesis by isolated chloroplasts was inhibited 40–85% by thiocarbamates, substituted ureas, and bromacil, but not by terbacil. The inhibitory effect of the urea derivatives was reversible, but that of thiocarbamates was irreversible. sn-Glycerol-3-phosphate acyltransferase(s) of the chloroplast and microsomal fractions were profoundly inhibited by thiocarbamates, but not by the other two groups of herbicides. Phosphatidic acid phosphatase was insensitive to all the herbicides tested.

Synthesis of Trilaurin by Developing Pisa Seeds (Actinodaphne hookeri)

The developing seeds of Actinodaphne hookeri were investigated to delineate their ability to synthesize large amounts of trilaurin. Until 88 days after flowering the embryos contained 71% neutral lipids (NL) and 29% phospholipids (PL) and both these components contained C-16:0, C-18:0, C-18:2, and C-18:3 as the major fatty acids (FA). At 102 days after flowering the seeds began to accumulate triacylglycerols (TAG) and to synthesize lauric acid (C-12:0). By 165 days after flowering, when the seeds were mature, they contained about 99% NL and 1% FL. At this stage the TAG contained exclusively C-12:0, while the PL consisted of long-chain fatty acids (LCFA) only. Leaf lipids in contrast did not contain any C-12:0. Experiments on [1-C-14]acetate incorporation into developing seed slices showed that at 88 days after flowering only 4% of the label was in TAG, 1% in diacylglycerols (DAG), and 87% in FL. One hundred two days after flowering seeds incorporated only 2% of the label into TAG, 30% into DAG, and 64% into FL. In contrast at 114 days after flowering 71% of the label was incorporated into TAG, 25% into DAG, and only 2% into FL. Analysis of labeled FA revealed that up to 102 days after flowering it was incorporated only into LCFA, whereas at 114 days after flowering it was incorporated exclusively into C-12:0. Furthermore, 67% of the label in PL at 114 days after flowering was found to be dilaurylglycerophosphate. Analysis of the label in DAG at this stage showed that it was essentially in dilaurin species. These observations indicate the induction of enzymes of Kennedy pathway for the specific synthesis of trilaurin at about 114 days after flowering, Homogenates of seeds (114 days after flowering) incubated with labeled FA in the presence of glycerol-3-phosphate and coenzymes A and ATP incorporated 84% of C-12:0 and 61% of C-14:0, but not C-16:0, C-18:2, and C-18:3, into TAG. In contrast the LCFA were incorporated preferentially into FL. It is concluded that, between 102 and 114 days after flowering, a switch occurs in A. hookeri for the synthesis of C-12:0 and trilaurin which is tissue specific. Since the seed synthesizes exclusively C-12:0 at 114 days after flowering onwards and incorporates specifically into TAG, this system appears to be ideal for identifying the enzymes responsible for medium-chain fatty acid as well as trilaurin synthesis and for exploiting them for genetic engineering. (C) 1994 Academic Press, Inc.

Oleosin Is Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Phospholipase Activities

In plants, fatty oils are generally stored in spherical intracellular organelles referred to as oleosomes that are covered by proteins such as oleosin. Seeds with high oil content have more oleosin than those with low oil content. However, the exact role of oleosin in oil accumulation is thus far unclear. Here, we report the isolation of a catalytically active 14 S multiprotein complex capable of acylating monoacylglycerol from the microsomal membranes of developing peanut cotyledons. Microsomal membranes from immature peanut seeds were solubilized using 8 M urea and 10 mM CHAPS. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 27 proteins in the 14 S complex. The major proteins present in the 14 S complex are conarachin, the major allergen Ara h 1, and other seed storage proteins. We identified oleosin 3 as a part of the 14 S complex, which is capable of acylating monoacylglycerol. The recombinant OLE3 microsomes from Saccharomyces cerevisiae have been shown to have both a monoacylglycerol acyltransferase and a phospholipase A(2) activity. Overexpression of the oleosin 3 (OLE3) gene in S. cerevisiae resulted in an increased accumulation of diacylglycerols and triacylglycerols and decreased phospholipids. These findings provide a direct role for a structural protein (OLE3) in the biosynthesis and mobilization of plant oils.

Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions

Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A(2) activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A(2) activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation.

Phospholipid Mediated Activation of Calcium Dependent Protein Kinase 1 (CaCDPK1) from Chickpea: A New Paradigm of Regulation

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V-max of the enzyme activity by these phospholipids significantly decreased the K-m indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K-1/2 = 114 nM) compared to PA (K-1/2 = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

Selective localisation of multi walled carbon nanotubes in polypropylene/natural rubber blends to reduce the percolation threshold

Polypropylene and natural rubber blends with multiwalled carbon nanotube (PP/NR + MWCNT nanocomposites) were prepared by melt mixing. The melt rheological behaviour of neat PP and PP/NR blends filled with different loadings (1, 3, 5, 7 wt%) of MWCNT was studied. The effect of PP/NR blends (with compositions, 80/20,50/50, 20/80 by wt) on the rheological percolation threshold was investigated. It was found that blending PP with NR (80/20 and 50/50 composition) reduced the rheological percolation threshold from 5 wt% to 3 wt% MWCNT. The melt rheological behaviour of the MWCNT filled PP/NR blends was correlated with the morphology observations from high resolution transmission electron microscopic (HRTEM) images. In predicting the thermodynamically favoured location of MWCNT in PP/NR blend, the specific interaction of phospholipids in NR phase with MWCNTs was considered quantitatively. The MWCNTs were selectively localised in the NR phase. The percolation mechanism in MWCNT filled PP/NR blends was discussed and for each blend composition, the percolation mechanism was found to be different. (C) 2015 Elsevier Ltd. All rights reserved.