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David T. Dennis - One of the best experts on this subject based on the ideXlab platform.
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Characterization and functional expression in yeast of a cDNA encoding NADP-dependent malic enzyme from castor oil seed
Botany, 2005Co-Authors: Heather L. Shearer, David T. DennisAbstract:A cDNA encoding NADP-dependent malic enzyme was isolated from castor bean (Ricinus communis L.) endosperm. The 2362-bp cDNA encodes a 641 amino acid protein, which is predicted to have a 50 amino acid chloroplast transit peptide. Southern blotting revealed a single copy of the gene. Recombinant protein expression confirmed that the cDNA encodes a NADP-dependent malic enzyme; both Western blotting and enzyme assays detected NADP-dependent malic enzyme. Removal of the transit peptide in the recombinant protein did not affect the pH optimum of the enzyme, but it did result in an increased specific activity.Key words: Ricinus communis, NADP-dependent malic enzyme (NADP-ME), fatty acid biosynthesis, Leucoplast, recombinant protein expression, transit peptide.
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Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm
Archives of biochemistry and biophysics, 2004Co-Authors: Heather L. Shearer, David H. Turpin, David T. DennisAbstract:Metabolic pathways sequestered within the Leucoplast of developing oilseeds ensure a balanced supply of substrates and cofactors for fatty acid biosynthesis. NADP-dependent malic enzyme (NADP-ME) may be important in supplying both carbon and NADPH for fatty acid biosynthesis in the developing endosperm of the oilseed Ricinus communis. NADP-ME was purified 5160-fold to a specific activity of 18.2 U/mg protein. NADP-ME is a homotetramer with a native mass of 254 kDa and a subunit size of approximately 63 kDa. Effectors of castor NADP-ME are typical of the NADP-malic enzymes, with the exception of acetyl-CoA and its derivatives, which were found to act as activators. This is consistent with a regulatory role for these molecules during fatty acid biosynthesis in vivo. NADP-ME was found to have maximal activity at stage 7 of endosperm development, coincident with maximal lipid accumulation.
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evidence that a malate inorganic phosphate exchange translocator imports carbon across the Leucoplast envelope for fatty acid synthesis in developing castor seed endosperm
Plant Physiology, 1997Co-Authors: Peter J Eastmond, David T. Dennis, Stephen RawsthorneAbstract:In this study we examined the processes by which malate and pyruvate are taken up across the Leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated Leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by Leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated Leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that Leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.
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Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm.
Plant physiology, 1997Co-Authors: Peter J Eastmond, David T. Dennis, Stephen RawsthorneAbstract:In this study we examined the processes by which malate and pyruvate are taken up across the Leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated Leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by Leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated Leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that Leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.
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Transit Peptides Play a Major Role in the Preferential Import of Proteins into Leucoplasts and Chloroplasts
The Journal of biological chemistry, 1996Co-Authors: Jiangxin Wan, Stephen D. Blakeley, David T. DennisAbstract:The in vitro import characteristics of six different precursors of plastid proteins were assessed to determine differences in the protein import pathways of Leucoplasts and chloroplasts. Five of these precursor proteins are destined to different subchloroplast sites, and one is a Leucoplast stromal precursor protein. The results indicate that some of these precursors can be imported equally into both plastid types and others preferentially into one type of plastid versus the other. The ability of plastids to import different proteins correlates with the in vivo steady state levels of these proteins. Additional differences were also observed in the intraorganellar portion of the translocation pathway for two thylakoidal proteins. The differences in import characteristics were found to be predominantly governed by information in the transit peptides, since attachment of the various transit peptides to different plastid and foreign proteins demonstrated that the import behavior of the proteins is transferable with the transit sequence. These results indicate that the import mechanisms of Leucoplasts and chloroplasts are sufficiently different such that the plastids respond differently to the information present in the transit peptides.
William C. Plaxton - One of the best experts on this subject based on the ideXlab platform.
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Leucoplast Isolation and Subfractionation
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: William C. PlaxtonAbstract:Leucoplasts are colorless plastids of nonphotosynthetic plant tissues that support a variety of anabolic roles, particularly the biosynthesis of long-chain fatty acids in storage tissues of developing oil seeds. They also perform other important metabolic functions including the biosynthesis of amino acids and tetrapyrrole compounds. Leucoplasts use a complex set of membrane carriers and channels to actively translocate nuclear-encoded precursor proteins from the cytosol, while exchanging various metabolites with the cytosol. Leucoplast purification is a necessary prerequisite for detailed studies of their soluble (stromal) and membrane (envelope) (phospho)proteomes, as well as for achieving a detailed understanding of their metabolic capabilities, transport processes, and biogenesis. This chapter describes protocols for Leucoplast purification from endosperm of developing castor oil seeds, and their subsequent subfractionation into envelope membrane and soluble stromal compartments for biochemical analysis.
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Molecular and regulatory properties of Leucoplast pyruvate kinase from Brassica napus (rapeseed) suspension cells.
Archives of biochemistry and biophysics, 2002Co-Authors: William C. Plaxton, Christopher R. Smith, Vicki L. KnowlesAbstract:Plastidic pyruvate kinase (PK(p)) from Brassica napus suspension cells was purified 431-fold to a final specific activity of 28 micromol phosphoenolpyruvate (PEP) utilized/min/mg protein. SDS-PAGE, immunoblot and gel filtration analyses indicated that this PK(p) exists as a 380-kDa heterohexamer composed of equal proportions of 64- (alpha-subunit) and 58-kDa (beta-subunit) polypeptides. The N-terminal sequence of the PK(p) alpha- and beta-subunits exhibited maximal identity with the corresponding regions deduced from putative PK genes of Arabidopsis thaliana and Methylobacterium extorquens, respectively. B. napus PK(p) displayed a sharp pH optimum of pH 8.0, and hyperbolic saturation kinetics with PEP and ADP (K(m) = 0.052 and 0.14 mM, respectively). 6-Phosphogluconate functioned as an activator (K(a) = 0.12 mM) by increasing V(max) by approximately 35% while decreasing the K(m)(PEP) and K(m)(ADP) values by 40 and 50%, respectively. 2-Oxoglutarate and oxalate were the most effective inhibitors (I(50) = 8.3 and 0.23 mM, respectively). A model is presented which highlights the role of 6-phosphogluconate in coordinating stromal NADPH and ATP production for anabolic processes of B. napus Leucoplasts.
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Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase
Plant physiology, 1991Co-Authors: William C. PlaxtonAbstract:Leucoplast pyruvate kinase (PKp; EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4°C. Purified castor seed PKp was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PKp) immunoglobulin G. Proteolytic activity against PKp was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4°C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PKp. The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nα-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PKp. This endopeptidase is present throughout all stages of endosperm development. Its PKp-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified Leucoplasts were incubated at 4°C for up to 21 hours, no degradation of PKp was observed; this indicated an extra-Leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PKp remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PKp.
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Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources.
Plant physiology, 1991Co-Authors: Stephen D. Blakeley, William C. Plaxton, David T. DennisAbstract:Two cDNA clones, PKpα and PKpβ, for the Leucoplast isozyme of pyruvate kinase have been isolated and characterized. A Southern blot of castor (Ricinus communis) DNA probed with PKpα indicates the presence of a single gene for PKp. Most (1610 base pairs) of the sequence of both cDNAs is identical. These 1610 base pairs begin with an ATG translation initiation codon, and have 248 base pairs of 3′-untranslated and 1362 base pairs of coding sequence. The sequences of the two clones 5′- to the identical regions are different but both encode peptides with a high percentage of hydrophobic amino acids. The derived sequence of PKpα encodes eight amino acid residues which have been identified as the amino-terminus of one subunit of PKp from castor seed Leucoplasts when the enzyme is purified in the absence of cysteine endopeptidase inhibitors. The sequence upstream of these amino acids is possibly the transit peptide for this protein. When PKp is extracted under conditions that eliminate its proteolytic degradation, its α-subunit has a relative molecular weight equal to the full-length coding sequence of PKpα. The data indicate that the transit peptide for the subunit of Leucoplast pyruvate kinase encoded by PKpα is not cleaved until the protein is released from the plastid. The derived amino acid sequences of PKpα and PKpβ are most closely related to Escherichia coli pyruvate kinase. Although the residues involved in substrate binding are conserved in Leucoplast pyruvate kinase, there is no phosphorylation site and only 5 of 15 amino acids in the E. coli fructose-1,6-bisphosphate binding site are conserved.
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Leucoplast Pyruvate Kinase fromDeveloping Castor OilSeeds1 Characterization oftheEnzyme's Degradation byaCysteine Endopeptidase
1991Co-Authors: William C. PlaxtonAbstract:Leucoplast pyruvate kinase (PKp; EC2.7.1.40) fromendosperm ofdeveloping castor oilseeds(Ricinus communis L.cvBaker 296) appears tobehighly susceptible tolimited degradation bya cysteine endopeptidase during thepurification oftheenzymeor incubation ofclarified homogenates at40C. Purified castor seed PKpwaspreviously reported toconsist ofimmunologically related 57.5and44kilodalton subunits (Plaxton WC,Dennis DT,Knowles VL[1990] Plant Physiol 94:1528-1534). Bycontrast, immunoreactive polypeptides ofabout63.5and54kilodaltons were observed whena western blotofanextract prepared under denaturing conditions wasprobed withaffinity purified rabbit anti(castor seedPKp)immunoglobulin G.Proteolytic activity against PKpwasestimated bythedisappearance ofthe63.5and54 kilodalton subunits andtheconcomitant appearance oflower molecular massimmunoreactive degradation products during the incubation ofclarified homogenates at40C.Thepresence of2 millimolar dithiothreitol accelerated thedegradation ofPKp. The conservation ofthe63.5 and54kilodalton subunits wasobserved after extraction oftheenzymeinthepresence of1millimolar phydroxymecuribenzoate, or1millimolar Na-p-tosyl-L-lysine
Stephen Rawsthorne - One of the best experts on this subject based on the ideXlab platform.
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evidence that a malate inorganic phosphate exchange translocator imports carbon across the Leucoplast envelope for fatty acid synthesis in developing castor seed endosperm
Plant Physiology, 1997Co-Authors: Peter J Eastmond, David T. Dennis, Stephen RawsthorneAbstract:In this study we examined the processes by which malate and pyruvate are taken up across the Leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated Leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by Leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated Leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that Leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.
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Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm.
Plant physiology, 1997Co-Authors: Peter J Eastmond, David T. Dennis, Stephen RawsthorneAbstract:In this study we examined the processes by which malate and pyruvate are taken up across the Leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated Leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by Leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated Leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that Leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.
David H. Turpin - One of the best experts on this subject based on the ideXlab platform.
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Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm
Archives of biochemistry and biophysics, 2004Co-Authors: Heather L. Shearer, David H. Turpin, David T. DennisAbstract:Metabolic pathways sequestered within the Leucoplast of developing oilseeds ensure a balanced supply of substrates and cofactors for fatty acid biosynthesis. NADP-dependent malic enzyme (NADP-ME) may be important in supplying both carbon and NADPH for fatty acid biosynthesis in the developing endosperm of the oilseed Ricinus communis. NADP-ME was purified 5160-fold to a specific activity of 18.2 U/mg protein. NADP-ME is a homotetramer with a native mass of 254 kDa and a subunit size of approximately 63 kDa. Effectors of castor NADP-ME are typical of the NADP-malic enzymes, with the exception of acetyl-CoA and its derivatives, which were found to act as activators. This is consistent with a regulatory role for these molecules during fatty acid biosynthesis in vivo. NADP-ME was found to have maximal activity at stage 7 of endosperm development, coincident with maximal lipid accumulation.
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Malate- and Pyruvate-Dependent Fatty Acid Synthesis in Leucoplasts from Developing Castor Endosperm
Plant Physiology, 1992Co-Authors: Ronald G. Smith, David A. Gauthier, David T. Dennis, David H. TurpinAbstract:Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoll gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to Leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by Leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, Leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.
Peter J Eastmond - One of the best experts on this subject based on the ideXlab platform.
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evidence that a malate inorganic phosphate exchange translocator imports carbon across the Leucoplast envelope for fatty acid synthesis in developing castor seed endosperm
Plant Physiology, 1997Co-Authors: Peter J Eastmond, David T. Dennis, Stephen RawsthorneAbstract:In this study we examined the processes by which malate and pyruvate are taken up across the Leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated Leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by Leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated Leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that Leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.
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Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm.
Plant physiology, 1997Co-Authors: Peter J Eastmond, David T. Dennis, Stephen RawsthorneAbstract:In this study we examined the processes by which malate and pyruvate are taken up across the Leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated Leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by Leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated Leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that Leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.