Pathways of Fatty Acid hydroperoxide metabolism in spinach leaf chloroplasts

The metabolism of 13-hydroperoxylinolenic acid was examined in protoplasts and homogenates prepared from mature leaves of spinach (Spinacia oleracea L.). Chloroplast membranes were the principal site for metabolism of the compound by at least two highly hydrophobic enzyme systems, hydroperoxide lyase and hydroperoxide dehydrase, the new name for an enzyme system formerly known as hydroperoxide isomerase and hydroperoxide cyclase. Hydroperoxide lyase was most active above pH 7 and could be separated from hydroperoxide dehydrase by anion exchange chromatography. Hydroperoxide dehydrase, measured by the formation of both α-ketol product and 12-oxo-phytodienoic acid, had its optimum activity in the range of pH 5 to 7. Lyase was more active than dehydrase activity when the enzymes were extracted by homogenization. The reverse was true when the enzyme activities were measured in protoplasts, which are isolated by gentle extraction methods. The variation in enzyme activity ratios with extraction methods suggests that hydroperoxide lyase is activated by plant injury and thus may function in a wound response. In the absence of injury, the normal pathway of fatty acid hydroperoxide metabolism is probably by hydroperoxide dehydrase activity. The molecular weights of both the lyase and dehydrase were approximately 220,000, as estimated by gel filtration.     

On the light dependence of Fatty Acid synthesis in spinach chloroplasts

The capacity of intact chloroplasts to synthesize long chain fatty acids from acetate depends on the stroma pH in Spinacia oleracea, U. S. hybrid 424. The pH optimum is close to 8.5. Lowering of the stroma pH leads to a reduction of acetate incorporation but does not suffice to eliminate fatty acid synthesis completely. Chain elongation from palmitic to oleic acid shows the same pH dependence. Fatty acid synthesis is activated in the dark upon the simultaneous addition of dihydroxyacetone phosphate and orthophosphate supplying ATP and oxaloacetate for reoxidation of NADPH in the stroma. Under these conditions both dark fatty acid synthesis and synthesis of oleate from palmitate show the same pH dependence as in the light. Dark fatty acid synthesis is further stimulated by increasing the stromal Mg²⁺ concentration with the ionophore A 23187. In contrast to CO₂ fixation, dark fatty acid synthesis is considerably reduced by dithiothreitol (DTT). This observation may be due to an acetyl-CoA deficiency, caused by a nonenzymic acylation of DTT, and a competition for ATP between DTT-activated CO₂ fixation and fatty acid synthesis. Because d,l-glyceraldehyde as inhibitor of CO₂ fixation compensates the DTT effect on dark fatty acid synthesis, reducing equivalents may be involved in the light dependence of acetate activation.     

Characterization of Fatty Acid biosynthesis in isolated pea root plastids

Fatty acid biosynthesis from Na[1-¹⁴C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl₂, 1 millimolar each of MnCl₂ and glycerol-3-phosphate, 15 millimolar KHCO₃, 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35°C. At the standard incubation temperature of 25°C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [¹⁴C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.     

Shikimate kinase from spinach chloroplasts : purification, characterization, and regulatory function in aromatic amino Acid biosynthesis

Shikimate kinase was purified to near homogenity from spinach Spinacia oleracea L. chloroplasts and found to consist of a single 31 kilodalton polypeptide. The purified enzyme was unstable, but could be stabilized by a variety of added proteins, including oxidized and reduced thioredoxins. Whereas the isolated enzyme was stimulated by mono- and dithiol reagents, the enzyme in intact chloroplasts was unaffected by added thiols and showed only minor response to dark/light transitions. These results indicate that the previously reported stimulation of shikimate kinase activity by reduced thioredoxins is due to enzyme stabilization rather than to activation. In the current study, the purified enzyme was inhibited by added ADP and showed a strong response to energy charge. When intact chloroplasts were incubated in the dark in presence of shikimate, phosphoenolpyruvate and a source of ATP (dihydroxyacetone phosphate or ATP itself under appropriate conditions), aromatic amino acids were formed: phenylalanine and tyrosine. The data indicate that energy charge plays a role in regulating shikimate kinase, thereby controlling the shikimate pathway. An unidentified enzyme of the latter part of the pathway, leading from shikimate-3-phosphate to phenylalanine, appears to be activated by light.     

A preliminary analysis of Fatty Acid synthesis in pea roots

Subcellular fractions from pea (Pisum sativum L.) roots have been prepared by differential centrifugation techniques. Greater than 50% of the recovered plastids can be isolated by centrifugation at 500g for 5 minutes. Plastids of this fraction are largely free from mitochondrial and microsomal contamination as judged by marker enzyme analysis. De novo fatty acid biosynthesis in pea roots occurs in the plastids. Isolated pea root plastids are capable of fatty acid synthesis from acetate at rates up to 4.3 nanomoles per hour per milligram protein. ATP, bicarbonate, and either Mg²⁺ or Mn²⁺ are all absolutely required for activity. Coenzyme A at 0.5 millimolar improved activity by 60%. Reduced nucleotides were not essential but activity was greatest in the presence of 0.5 millimolar of both NADH and NADPH. The addition of 0.5 millimolar glycerol-3-phosphate increased activity by 25%. The in vitro and in vivo products of fatty acid synthesis from acetate were primarily palmitate, stearate, and oleate, the proportions of which were dependent on experimental treatments. Fatty acids synthesized by pea root plastids were recovered in primarily phosphatidic acid and diacylglycerol or as water soluble derivatives and the free acids. Lesser amounts were found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and monogalactosyldiacylglycerol.     

Role of plastidial acyl-acyl carrier protein: Glycerol 3-phosphate acyltransferase and acyl-acyl carrier protein hydrolase in channelling the acyl flux through the prokaryotic and eukaryotic pathway

Monoclonal antibodies raised against extracts of the rachis abscission zone of Sambucus nigra L. were selected for high reactivity towards abscission-zone proteins. One antibody (YZ1/2.23) has been shown to cross-react, by both indirect and competition enzyme-linked immunosorbent assay and by Western blotting, with a number of plant enzymes including horseradish peroxidase, rice -glucosidase, almond -glucosidase and the lectins from Phaseolus vulgaris and Erythrina cristagalli.The major N-linked oligosaccharide isolated from horseradish peroxidase has the sequence Man 3(Man6)(Xyl2)Man4GlcNAc4(Fuc3) GlcNAc. This oligosaccharide was found to be a potent inhibitor of the binding of YZ1/2.23 to the intact glycoprotein. The common determinant is therefore contained within this structure.Abbreviations ELISA enzyme-linked immunosorbent assay - Fuc fucose - GlcNAc N-acetylglucosamine - HRP horseradish peroxidase - Ig immunoglobulin - Man mannose - SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Xyl xylose     

Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed

A cDNA for a structurally variant acyl-acyl carrier protein (ACP) desaturase was isolated from milkweed (Asclepias syriaca) seed, a tissue enriched in palmitoleic (16:1⁹)* and cis-vaccenic (18:1¹¹) acids. Extracts of Escherichia coli that express the milkweed cDNA catalyzed ⁹ desaturation of acyl-ACP substrates, and the recombinant enzyme exhibited seven- to ten-fold greater specificity for palmitoyl (16:0)-ACP and 30-fold greater specificity for myristoyl (14:0)-ACP than did known ⁹-stearoyl (18:0)-ACP desaturases. Like other variant acyl-ACP desaturases reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously characterized ⁹-18:0-ACP desaturases. Based on the activity of an N-terminal deletion mutant of a⁹ -18:0-ACP desaturase, this structural feature likely does not account for differences in substrate specificities.     

Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl-acyl carrier protein desaturase and ferredoxin.

Expression of a plant delta 6-palmitoyl (16:0)-acyl carrier protein desaturase in Escherichia coli resulted in the accumulation of the novel monounsaturated fatty acids delta 6-hexadecenoic acid (16:1 delta 6) and delta 8-octadecenoic acid. Amounts of 16:1 delta 6 accumulated by E. coli were increased more than twofold by the expression of a plant ferredoxin together with the delta 6-16:0-acyl carrier protein desaturase.     

Fat metabolism in higher plants. Production of short- and medium-chain acyl-acyl carrier protein by spinach stroma preparations treated with cerulenin.

Incubation of stroma preparations from spinach chloroplasts with low concentrations of cerulenin (10 muM) resulted in severe inhibition of fatty acid synthesis but stimulated the release of medium-chain acids in very high proportions (60-70%). Preincubation of these preparations with cerulenin in the absence of substrate exerted no additional effect on subsequent fatty acid synthesis (as measured by incorporation of [14C]acetate into fatty acids) or the pattern of radioactive acids obtained. Acyl-protein, acyl-CoA, free fatty acids and lipids were resolved from each other and analysed for their distribution of 14C-labelled fatty acids. Acyl-protein derived from cerulenin-treated preparations was the only fraction which contained short- and medium-chain acids (C6--C12). The other fractions from both control and cerulenin-treated groups consisted exclusively of C16 and C18 acids. Acyl-protein was purified by gel filtration chromatography and was characterized as acyl-acyl carrier protein.     

In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis

In order to examine potential regulatory steps in plant fatty acid biosynthesis, we have developed procedures for the analysis of the major acyl-acyl carrier protein (ACP) intermediates of this pathway. These techniques have been used to separate and identify acyl-ACPs with chain configurations ranging from 2:0 to 18:1 and to determine the relative in vivo concentrations of acyl-ACPs in spinach leaf and developing seed. In both leaf and seed as much as 60% of the total ACPs were nonesterified (free), with the remaining proportion consisting of acyl-ACP intermediates leading to the formation of palmitate, stearate, and oleate. In spinach leaf the proportions of the various acyl groups esterified to each ACP isoform were indistinguishable, indicating that these isoforms are utilized similarly in de novo fatty acid biosynthesis in vivo. However, the acyl group distribution pattern of seed ACP-II differed significantly from that of leaf ACP-II. The malonyl-ACP levels were less than the 4:0-ACP and 6:0-ACP levels in leaf, and in contrast, the malonyl-ACP-II levels in seed were approximately 3-fold higher than the 4:0-ACP-II and 6:0-ACP-II levels. In addition, the ratio of oleoyl-ACP-II (18:1) to stearoyl-ACP-II (18:0) was higher in seed than in leaf. These data suggest that the differences in acyl-ACP patterns reflect a tissue/organ-specific difference rather than an isoform-specific difference. In extracts prepared from leaf samples collected in the dark, the levels of acetyl-ACPs were approximately 5-fold higher compared to samples collected in the light. The levels of free ACPs showed an inverse response, increasing in the light and decreasing in the dark. Notably there was no concomitant increase in the malonyl-ACP levels. The most likely explanation for the major increase in acetyl-ACP levels in the dark is that light/dark control over the rate of fatty acid biosynthesis occurs at the reaction catalyzed by acetyl-CoA carboxylase.     

The formation of homogentisate in the biosynthesis of tocopherol and plastoquinone in spinach chloroplasts

Homogentisate is the precursor in the biosynthesis of -tocopherol and plastoquinone-9 in chloroplasts. It is formed of 4-hydroxyphenylpyruvate of the shikimate pathway by the 4-hydroxyphenylpyruvate dioxygenase. In experiments with spinach the dioxygenase was shown to be localized predominatedly in the chloroplasts. Envelope membranes exhibit the highest specific activity, however, because of the high stromal portion of chloroplasts, 60–80% of the total activity is housed in the stroma. The incorporation of 4-hydroxyphenylpyruvate into 2-methyl-6-phytylquinol as the first intermediate in the tocopherol synthesis by the two-step-reaction: 4-Hydroxyphenylpyruvate Homogentisate 2-Methyl-6-phytylquinol was demonstrated by using envelope membranes. Homogentisate originates directly from 4-hydroxyphenylpyruvate of the shikimate pathway. Additionally, a bypass exists in chloroplasts which forms 4-hydroxyphenylpyruvate from tyrosine by an L-amino-acid oxidase of the thylakoids and in peroxisomes by a transaminase reaction. Former results about the dioxygenase in peroxisomes were verified.     

Nucleoid proteins of pea chloroplasts: detection of a protein homologous to ribosomal protein.

Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction. Using RP-HPLC, the component composition of the basic proteins was studied. SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol. masses of components from 17 to 30 kDa. One polypeptide with mol. mass of 28 kDa (P28) was obtained by RP-HPLC. The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae. The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed.     

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.

The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.     

Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator

Using an 5-AvaII fragment of the spinach (Spinacia oleracea L.) phosphate translocator cDNA as a probe for a hybridization screening of a pea (Pisum sativum L.) cDNA library we have cloned and sequenced a cDNA clone coding for the phosphate translocator precursor protein from pea chloroplasts. The full-length cDNA clone comprises 42 base pairs (bp) at the 5-non-coding region, a 1206-bp coding region corresponding to a polypeptide of 402 amino-acid residues (relative molecular mass 43 671) and 244 bp at the non-coding 3-region. Determination of the N-terminal sequence of the phosphate translocator from both pea and spinach chloroplasts revealed that the transit peptides consist of 72 and 80 amino-acid residues, respectively. These transit peptides are different from those of other chloroplastic transit peptides in that they both contain an amphiphilic -helix which is located either in close proximity to the processing site in pea or at the N-terminus in spinach. The mature proteins from pea and spinach both contain about 87% identical amino-acid residues and about seven putative membrane-spanning -helices. Some of these -helices have an amphiphilic character and might serve to form a hydrophilic translocation channel through the membrane. The in-vitro synthesized pea precursor protein is directed to the chloroplast and inserted into the chloroplast envelope membrane.Abbreviations bp base pairs - kDa kilodaltons - M_r relative moleculas mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We wish to thank Dr D. Pappin and R. Jakes (AFRC Sequencing Laboratory, Department of Biochemistry, University of Leeds, UK) for performing the N-terminal sequence determinations and are greatful to Dr J. S. Gantt (Botany Department, University of Georgia, Athens, USA) for a pea leaf cDNA library and to Professor J. C. Gray (University of Cambridge, Department of Botany, Cambridge, UK) for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Science and Engineering Research Council and the Royal Society. D.L.W. was the recipient of the Royal Society Rosenheim research fellowship and K.F. was supported by a fellowship from the Studienstiftung des deutschen Volkes.     

Evidence for a lack of phospholipid transfer protein in the stroma of spinach chloroplasts

The possible activity of phospholipid transfer protein in stroma extracts from spinach leaf has been investigated. Stroma, prepared from purified intact chloroplasts, was dialyzed and passed through various chromatography columns. None of the protein fractions eluted was able to stimulate the transfer of phosphatidylglycerol (PG) or phosphatidylcholine (PC) from liposomes to mitochondria, suggesting the lack of phospholipid transfer protein in the stroma from mature spinach chloroplasts.     

哈氏弧菌脂酰-ACP合成酶的体外功能

【目的】系统鉴定哈氏弧菌脂酰-ACP合成酶(Acyl-ACP synthetase,Aas S)以不同链长游离脂肪酸和非脂肪链羧酸作为底物的体外催化反应。【方法】利用非变性蛋白质凝胶电泳和紫外分光光度计法从定性和定量两个方面分析了Aas S的体外催化功能与活性。【结果】Aas S能够催化不同链长直链的自由脂肪酸合成脂酰-ACP,其中以C6–C12作为底物时活性最高;以羟基脂肪酸作为底物的情况下,Aas S催化C8–C14的羟基脂肪酸有较高的活性。非脂肪链羧酸类作为底物的反应中,20种蛋白质氨基酸、苯甲酸和水杨酸均可以作为Aas S的底物,合成相应的脂酰-ACP。【结论】本研究系统地证明了哈氏弧菌脂酰-ACP合成酶(Aas S)对不同底物的不同催化活性,为生物体内氨基酸代谢和菌黄素合成代谢的研究提供了可行性的分析依据。