Structural characterization of alpha-terminal group of natural rubber. 1. Decomposition of branch-points by lipase and phosphatase treatments

Deproteinized natural rubber latex (DPNR-latex) was treated with lipase and phosphatase in order to analyze the structure of the chain-end group (alpha-terminal). The enzymatic treatment decreased the content of long-chain fatty acid ester groups in DPNR from about 6 to 2 mol per rubber molecule. The molecular weight and intrinsic viscosity were reduced to about one-third after treatment with lipase and phosphatase. The Huggins' k' constant of the enzyme-treated DPNR showed the formation of linear rubber molecules. The molecular weight distribution of DPNR changed apparently after treatment with lipase and phosphatase. (1)H NMR spectrum of rubber obtained from DPNR-latex showed small signals due to monophosphate, di-phosphate and phospholipids at the alpha-terminus. Treatment of DPNR-latex with lipase and phosphatase decreased the relative intensity of the (1)H NMR signals corresponding to phospholipids, whereas no change was observed for the signals due to mono- and diphosphates. The residual mono- and diphosphate signals as well as some phospholipid signals after lipase and phosphatase treatments indicate that mono- and diphosphate groups are directly linked at the alpha-terminus with the modified structure, expected by aggregation or linking with phospholipid molecules.     

Structural characterization of rubber from jackfruit and euphorbia as a model of natural rubber

A structural study of low molecular weight rubbers from Jackfruit (Artocarpus heterophyllus) and Painted spurge (Euphorbia heterophylla) was carried out as model compounds of natural rubber from Hevea brasiliensis. The rubber content of latex from Jackfruit was 0.4-0.7%, which is very low compared with that of 30-35% in the latex from Hevea tree. The rubber from Jackfruit latex was low molecular weight with narrow unimodal molecular weight distribution (MWD), whereas that obtained from E. heterophylla showed very broad MWD. The 1H and 13C NMR analyses showed that Jackfruit rubber consists of a dimethylallyl group and two trans-isoprene units connected to a long sequence of cis-isoprene units. The alpha-terminal group of Jackfruit rubber was presumed to be composed of a phosphate group based on the presence of 1H NMR signal at 4.08 ppm corresponding to the terminal =CH-CH2OP group.     

Nonuniformity in natural rubber as revealed by small-angle neutron scattering, small-angle X-ray scattering, and atomic force microscopy

The microscopic structures of natural rubber (NR) and deproteinized NR (DPNR) were investigated by means of small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM). They were compared to those of isoprene rubber (IR), which is a synthetic analogue of NR in terms of chemical structure without any non-rubber components like proteins. Comparisons of the structure and mechanical properties of NR, DPNR, and IR lead to the following conclusions. (i) The well-known facts, for example, the outstanding green strength of NR and strain-induced crystallization, are due not much to the presence of proteins but to other components such as the presence of phospholipids and/or the higher stereoregularity of NR. It also became clear the naturally residing proteins accelerate the upturn of stress at low strain. The protein phases work as cross-linking sites and reinforcing fillers in the rubbery matrix. (ii) The microscopic structures of NR were successfully reproduced by SANS intensity functions consisting of squared-Lorentz and Lorentz functions, indicating the presence of inhomogeneities in bulk and thermal concentration fluctuations in swollen state, respectively. On the other hand, IR rubbers were homogeneous in bulk. (iii) The inhomogeneities in NR are assigned to protein aggregates of the order of 200 A or larger. Although these aggregates are larger in size as well as in volume fraction than those of cross-link inhomogeneities introduced by cross-linking, they are removed by deproteinization. (iv) Swelling of both NR and IR networks introduces gel-like concentration fluctuations whose mesh size is of the order of 20 A.     

Modification of membrane lipid: physical properties in relation to fatty acid structure.

Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.     

Renewable resources from wild sunflowers (Helianthus spp., asteraceae)

Twenty-eight taxa of Helianthus collected throughout the United States and grown in afield nursery were evaluated for yields of oil, polyphenol hydrocarbon, protein, and ash in above-ground biomass. Hydrocarbons were examined for the presence of rubber, gutta, and waxes. Rubber and gutta were analyzed for weight-average molecular weight (ìw) and molecular weight distribution (MWD). Helianthus ciliaris had the highest oil yield (3.7%) and was analyzed for yield of fatty acids and unsaponifiable matter. Most taxa had low polyphenol yields (<10%), with H. strumosus having the highest (13.9%). Helianthus salicifolius yielded the most hydrocarbon (1.6%) and H. ciliaris had the highest protein content (13.4%). Natural rubber was present in 13 species of wild sunflowers. Helianthus maximiliani had the lowest ìw (29.8 x 10³), while H. laevigatus had the highest (73.3 x 10³). The MWD of rubber from sunflowers were within the range of those for commercial rubbers. The lower molecular weight rubbers may have potential as plasticizing additives in commercial processing of synthetic polyisoprenes and as hydrocarbon feedstock for a synthetic petroleum industry.     

Cyclopropane fatty acid synthase of Escherichia coli. Stabilization, purification, and interaction with phospholipid vesicles.

The cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes the methylenation of the unsaturated moieties of phospholipids in a phospholipid bilayer. The methylene donor is S-adenosyl-L-methionine. The enzyme is loosely associated with the inner membrane of the bacterium and binds to and is stabilized by phospholipid vesicles. The enzyme has been purified over 500-fold by flotation with phospholipid vesicles and appears to be a monomeric protein having a molecular weight of about 90 000. The enzyme binds only to vesicles of phospholipids which contain either unsaturated or cyclopropane fatty acid moieties. CFA synthase is active on phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, the major phospholipids of E. coli, and also has some activity on phosphatidylcholine. The enzyme is equally active on phospholipid vesicles in the ordered or the disordered states of the lipid phase transition. Studies with a reagent that reacts only with the phosphatidylethanolamine molecules of the outer leaflet of a phospholipid bilayer indicate that CFA synthase reacts with phosphatidylethanolamine molecules of both the outer and the inner leaflets of phospholipid vesicles.     

Muscle fatty acid-binding protein

Muscle or heart fatty acid-binding protein is a low molecular weight protein that binds long-chain fatty acids in the cytosol of muscle tissues. The three-dimensional structure of the human, bovine and insect proteins are known, either via X-ray or NMR techniques. The folding of the protein closely resembles that of the other FABPs: ten anti-parallel beta-strands are arranged to form a clam shell, closed at one end by two alpha-helices. This arrangement allows the formation of an internal cavity where the fatty acid can be accommodated, protected and isolated from the external environment. The fatty acid in the protein interior is stabilized by electrostatic and hydrogen bond interactions of its carboxylic head with charged or polar residues of the protein and by interactions of its tail with hydrophobic residues. The three-dimensional structure of different fatty acid-protein complexes along with molecular dynamics simulations are now providing insight into the molecular details of the specificity of the ligand binding.     

The role of defensin fragment in the regulation of fatty acid composition of membrane phospholipids in epithelial-like cells

It is shown that a tetrapeptide fragment of defensin does not alter the phospholipid composition in the membranes of CHO-K1 cells but regulates the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine (PEA), phosphatidylserine (PS), and phosphatidylinositol (PI). Incubation of the cells in the presence of this tetrapeptide resulted in modification of unsaturated fatty acid composition in the studied phospholipids. The content of monoenoic (mainly C18 : 1ω9) and/or dienoic (C18 : 2ω6) fatty acids increased, while the level of polyenoic fatty acids decreased. It was found that in the polyenoic fatty acid group of the PEA, PS and PI molecules, the ω3-/ω6-acid ratio decreased mainly due to the lower content of long-chain ω3-acids with 20 and/or 22 carbonic atoms. The possible role of this peptide in inhibition of the activity of Δ6- and Δ5-desaturases involved in the synthesis of long-chain polyenoic fatty acids, the quantitative alteration of which in phospholipids influences physicochemical parameters in cell membranes, is discussed.     

Fatty-acid synthesis in lactating-goat mammary gland. 1. Medium-chain fatty-acid synthesis.

Tissue slices from lactating goat-mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long-chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle-free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short-chain and long-chain fatty acids. Terminating acyl-thioesterases of low molecular weight could not be detected in the particle-free supernatant. Addition of the microsomal fraction to the particle-free supernatant induced the synthesis of medium-chain fatty acids in proportions which were similar to those found in goat milk fat.     

Natural rubber biosynthesis in plants,the rubber transferase complex,and metabolic engineering progress and prospects

Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR‐enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)—the synthesis of the high molecular weight rubber polymer itself—is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.     

Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid

The formation of lysophosphatidylcholines from unsaturated phosphatidylcholines upon treatment with hypochlorous acid was evaluated by means of MALDI-TOF mass spectrometry and 31P NMR spectroscopy. With an increasing number of double bonds in a fatty acid residue, the yield of lysophosphatidylcholines with a saturated fatty acid residue increased considerably in comparison to the total amount of higher molecular weight products like chlorohydrins and glycols. High amounts of lysophosphatidylcholines were formed from phospholipids containing arachidonic or docosahexaenoic acid residues. In phospholipids with monounsaturated fatty acid residues, the position of the double bond did not influence the yield of lyso-products. Besides the exclusive formation of chlorohydrin and glycol, hypochlorous acid caused the cleavage of the unsaturated fatty acid residue independent of its location at the first or second position of the glycerol backbone. In contrast, strong alkaline conditions, i.e. saponification led also to a hydrolysis of the saturated fatty acid residue from phosphatidylcholines. It is concluded that both MALDI-TOF mass spectrometry and 31P NMR spectroscopy are able to detect the formation of lysophosphatidylcholines. We conclude also that the formation of lysophospholipids from unsaturated phosphatidylcholines by hypochlorous acid can be relevant in vivo under acute inflammatory conditions.     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.     

Short-chain lecithin/long-chain phospholipid unilamellar vesicles: asymmetry, dynamics, and enzymatic hydrolysis of the short-chain component

Asymmetric unilamellar vesicles are produced when short-chain phospholipids (fatty acyl chain lengths of 6-8 carbons) are mixed with long-chain phospholipids (fatty acyl chain lengths of 14 carbons or longer) in ratios of 1:4 short-chain/long-chain component. Short-chain lecithins are preferentially distributed on the outer monolayer, while a short-chain phosphatidylethanolamine derivative appears to localize on the inner monolayer of these spontaneously forming vesicles. Lanthanide NMR shift experiments clearly show a difference in head-group/ion interactions between the short-chain and long-chain species. Two-dimensional 1H NMR studies reveal efficient spin diffusion networks for the short-chain species embedded in the long-chain bilayer matrix. The short-chain lecithin is considerably more mobile than the long-chain component but has hindered motion compared to short-chain lecithin micelles. This differentiation in physical characteristics of the two phospholipid components is critical to understanding the activity of phospholipases toward these binary systems.     

Synthesis of a metabolically stable modified long-chain fatty acid salt and its photolabile derivative

An analogue of the long-chain fatty acid salt, sodium stearate, was synthesized in which the hydrogen atoms at carbons 2, 3, and 18 were replaced by fluorine. The key step in the synthesis was the addition of 3-iodo-2,2,3,3-tetrafluoropropanoic acid amide to 15,15,15-trifluoro-1-pentadecene. Radioactivity was introduced by catalytic reduction of 2,2,3,3,18,18,18-heptafluoro-4-octadecenoic acid amide with carrier-free tritium gas yielding a product with the specific radioactivity of 2.63 TBq/mmol. The resulting 2,2,3,3,18,18,18-heptafluoro-4-octadecenoic acid has a pKa of about 0.5 and is completely dissociated under normal physiological conditions. The fluorinated fatty acid salt analogue is readily taken up into hepatocytes and proved to be metabolically inert. In an approach to the identification of proteins involved in long-chain fatty acid salt transport across membranes and intracellular compartments, the photolabile derivative 11,11-azo-2,2,3,3,18,18,18-heptafluoro[G-3H]octadecanoic acid sodium salt was synthesized with a specific radioactivity of 2.63 TBq/mmol. Photolysis of the photolabile derivative, using a light source with a maximum emission at 350 nm, occurred with a half-life of 1.5 min. The generated carbene reacted with 14C-labeled methanol and acetonitrile with covalent bond formation of 6-13%. Its efficacy for photoaffinity labeling was demonstrated by incorporation into serum albumin, the extracellular fatty acid salt-binding protein, as well as into the intracellular fatty acid salt-binding protein (FABP) of rat liver with the molecular weight of 14,000.     

Digestion and absorption of polyunsaturated fatty acids.

Polyunsaturated fatty acids play an important part in the structure and function of cellular membranes and are precursors of lipid mediators which play a key role in cardiovascular and inflammatory diseases. Dietary sources of essential fatty acids are vegetable oils for either linoleic or alpha-linolenic acids, and sea fish oils for eicosapentaenoic and docosahexaenoic acids. Because of the specificity of the pancreatic lipid hydrolases, triglyceride fatty acid distribution is an essential parameter in the digestibility of fats. The efficiency of the intestinal uptake depends on the hydrolysis and especially on their micellarization. n-3 polyunsaturated fatty acid ethyl ester digestion is recognized to be impaired, but n-3 polyunsaturated fatty acid triglyceride hydrolysis remains a controversial point, and to some authors explains differences observed between vegetable and fish oil absorption. So additional studies are required to investigate this intestinal step. In enterocytes, morphological and biochemical absorption processes involve reesterification of long-chain fatty acids and lipoprotein formation. At this level, specific affinity of I- and L-FABPc (cytosolic fatty acid binding proteins) to polyunsaturated fatty acids requires further investigation. A better understanding of the role of these FABPc might bring to light the esterification step, particularly the integration of polyunsaturated fatty acids into phospholipids. With reference to differences published between fish and vegetable oil absorption, longer-term absorption studies appear essential to some authors. Polyunsaturated fatty acid absorption is thought to be not very dissimilar to that of long-chain mono-unsaturated fatty acid absorption. However, several digestion and absorption specific steps are worth studying with reference to the crucial role of polyunsaturated fatty acids in the organism, and for example adaptation of possible dietary supplements.     

The lipid composition of the electric organ of the ray, Torpedo marmorata, with specific reference to sulfatides and Na+-K+-ATPase.

The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.     

Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate

Rat intestinal fatty-acid-binding protein (I-FABP) is a small (15,124 Mr) cytoplasmic polypeptide that binds long-chain fatty acids in a non-covalent fashion. I-FABP is a member of a family of intracellular binding proteins that are thought to participate in the uptake, transport and/or metabolic targeting of hydrophobic ligands. The crystal structure of Escherichia coli-derived rat I-FABP with a single molecule of bound palmitate has been refined to 2 A resolution using a combination of least-squares methods, energy refinement and molecular dynamics. The combined methods resulted in a model with a crystallographic R-factor of 17.8% (7775 reflections, sigma greater than 2.0), root-mean-square bond length deviation of 0.009 A and root-mean-square bond angle deviation of 2.85 degrees. I-FABP contains ten antiparallel beta-strands organized into two approximately orthogonal, beta-sheets. The hydrocarbon tail of its single C16:0 ligand is present in a well-ordered, distinctively bent conformation. The carboxylate group of the fatty acid is located in the interior of I-FABP and forms a unique "quintet" of electrostatic interactions involving Arg106; Gln 115, and two solvent molecules. The hydrocarbon tail is bent with a slight left-handed helical twist from the carboxylate group to C-16. The bent methylene chain resides in a "cradle" formed by the side-chains of hydrophobic, mainly aromatic, amino acid residues. The refined molecular model of holo-I-FABP suggests several potential locations for entry and exiting of the fatty acid.     

Effects of two different medium-chain triglycerides (tri C8:O and tri C12:O) on liver lipids in the growing rat.

Results presented in this study emphasize the long-term effects of dietary fatty acid chain length on some biochemical parameters of the liver in the growing rat. High levels of medium-chain fatty acids feeding (C8:O and C12:O) from 40 to 340 g of body weight induced liver growth and lipid contents intermediary between values recorded with a lipid-free diet and with a diet containing long-chain fatty acids. No steatosis was recorded but neutral lipid contents appeared to be correlated with the dietary fatty acid chain length while phospholipid contents remained much more stable. In the four tested nutritional conditions, only dodecano?c or lauric acid (C12:O) feeding induced important alterations in the fatty acid pattern of liver neutral lipids. Medium-chain fatty acids, and even lauric acid which was intensely esterified in adipose tissue triglycerides, did not appreciably modify the fatty acid composition of liver phospholipids and were not esterified in it.     

酿酒酵母酚类抑制物耐受性脂质组学研究

通过脂质组学分析方法从细胞膜磷脂分布方面探究适应进化酿酒酵母酚酸耐受性机制。主要利用高效液相色谱-质谱(LC-MS)对酚酸胁迫下适应进化菌株和原始菌株脂质成分检测并进行统计学比较分析。检测出565种脂质代谢物,包含细胞膜磷脂185种。相比初始菌株,适应进化菌株细胞膜中磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)和磷脂酰肌醇(PI)类磷脂分子相对含量增加,含有长链(C32-C36)和双不饱和脂酰链的磷脂分子含量增加。统计学分析表明显著性差异磷脂分子主要为含有长链不饱和脂酰链的PC和PE类磷脂分子。推测适应进化菌株通过膜磷脂重塑提高细胞膜完整性,对酚类抑制物起到选择性屏障作用,从而保持细胞活性。