bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Characterization of Potato Fumarate Hydratase and Functional Expression in Escherichia coli
Authors: Nast G. , Muller-Rober B. 
Published: July 2002

Abstract
The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.
Purification of a Protein Phosphatase from Chloroplast Stroma Capable of Dephosphorylating the Light-Harvesting Complex-II
Authors: Hammer M. F. , Markwell J. , Sarath G. 
Published: July 2002

Abstract
A protein phosphatase was purified from the stroma of Pea (Pisum sativum L.) chloroplasts that is capable of dephosphorylating synthetic phosphopeptides. Following chromatographic purification of greater than 400-fold, two-dimensional electrophoresis indicated that the stromal protein phosphatase is a 29-kD protein. A similar molecular size was determined for the protein-phosphatase activity using gel-permeation chromatography, indicating that the stromal protein phosphatase is probably a monomer. The purified enzyme was able to dephosphorylate synthetic phosphopeptides, which mimic the thylakoid light-harvesting complex-II (LHC-II) N terminus, as well as LHC-II in thylakoid membranes, but did not dephosphorylate the major 64-kD phosphoprotein in the stroma. The stromal protein phosphatase did not discriminate between dephosphorylation of phosphothreonine and phosphoserine residues in synthetic peptide substrates, providing further evidence that this enzyme is distinct from the protein phosphatase localized in thylakoid membranes. The exact physiological role of the stromal protein phosphatase has yet to be determined, but it may function in the dephosphorylation of LHC-II.
Gibberellin Biosynthesis from Gibberellin A12-Aldehyde in Endosperm and Embryos of Marah macrocarpus
Authors: MacMillan J. , Ward D. A. , Phillips A. L. , Sanchez-Beltran M. J. , Gaskin P. , Lange T. , Hedden P. 
Published: July 2002

Abstract
Soluble enzyme preparations from embryos and endosperm of Marah macrocarpus (previously Echinocystis macrocarpa) were incubated with [14C4]gibberellin(GA)12-aldehyde,[14C4]GA12, [14C1] GA9, 2,3-didehydro[14C1]GA9, [14C1]GA20, and [17–13C,3H]GA5. Embryo preparations converted GA12-aldehyde, GA12, and GA9 to GA4 and GA7; 2,3-didehydroGA9 to GA7; GA5 to GA3; and GA20 (incompletely) to GA1 and GA60, but not to GA3. Endosperm preparations converted GA12-aldehyde and GA12 to GA15, GA24, GA25, and GA9, but, unlike embryo preparations, not to GA4 or GA7. However, GA4 and GA7 were formed from GA9 and GA7 was formed from 2,3-didehydroGA9. Metabolism of GA5 to GA3 and GA20 to GA1 was low. 2,3-DidehydroGA9 accumulated when GA9 was incubated with a desalted endosperm preparation. A cDNA clone (M3–8), selected from an embryo-derived cDNA library using a DNA fragment generated by reverse transcriptase polymerase chain reaction, was expressed in Escherichia coli. The fusion protein converted GA12 to GA9 (major) and GA25 (minor); GA53 was metabolized less effectively and only to GA44. Thus, the M3–8 protein is functionally similar to GA 20-oxidases from Arabidopsis thaliana, Spinacia oleracea, and Pisum sativum, but different from that from Cucurbita maxima seeds, to which its amino acid sequence is most closely related. mRNA hybridizing to M3–8 accumulated in embryos and endosperm of M. macrocarpus, but was absent in vegetative tissues.
A Defective Signal Peptide Tethers the floury-2 Zein to the Endoplasmic Reticulum Membrane
Authors: Gillikin J. W. , Zhang F. , Coleman C. E. , Bass H. W. , Larkins B. A. , Boston R. S. 
Published: July 2002

Abstract
The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD α--zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD α--zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this α--zein reveals a putative mutation alaine-&gt;valine (Ala-&gt;Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 α--zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 α--zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 α--zein and the fl2 α--zein (Val-&gt;Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 α--zein (Val-&gt;Ala) was localized in the soluble portion of the microsomes, whereas the fl2 α--zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.
Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA1
Authors: Andersen Scott E. , Bastola Dhundy R. , Minocha Subhash C. 
Published: July 2002

Abstract
The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [14C]Arg, [14C]Orn, and [14C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [14C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.
Two Pine Endo-β-1,4-Glucanases Are Associated with Rapidly Growing Reproductive Structures
Authors: Loopstra Carol A. , Mouradov Aidyn , Vivian-Smith Adam , Glassick Tina V. , Gale Beth V. , Southerton Simon G. , Marshall Heidi , Teasdale Robert D. 
Published: July 2002

Abstract
Two cDNA clones encoding endo-β-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (Pinusradiatacellulase 1) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.
Chlorophyll Synthesis in Dark-Grown Pine Primary Needles
Authors: Schoefs Benoı̂t , Franck Fabrice 
Published: July 2002

Abstract
The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllidea spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.
Methyl Jasmonate Induces Lauric Acid ω-Hydroxylase Activity and Accumulation of CYP94A1 Transcripts but Does Not Affect Epoxide Hydrolase Activities inVicia sativaSeedlings
Authors: Pinot Franck , Benveniste Irène , Salaün Jean-Pierre , Durst Francis 
Published: July 2002

Abstract
Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mm solution of MetJA stimulated ω-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min−1mg−1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid ω-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.
Occurrence of Cello-Oligosaccharides in the Apoplast of Auxin-Treated Pea Stems
Authors: Tominaga Rumi , Samejima Masahiro , Sakai Fukumi , Hayashi Takahisa 
Published: July 2002

Abstract
Treatment of pea (Pisum sativum L.) hypocotyl segments with indole-3-butyric acid, which promotes segment elongation, increased the solubilization of both xyloglucan and cello-oligosaccharides in the apoplast of auxin-treated pea stems. The cello-oligosaccharides were isolated from the apoplastic solution with a charcoal/Celite column and were identified as cellobiose, cellotriose, and cellotetraose after subsequent thin-layer chromatography and paper electrophoresis. Cello-oligosaccharides in the apoplastic fraction were monitored using cellobiose dehydrogenase. Both xyloglucan and cello-oligosaccharides appeared to be formed concurrently within 30 min after treatment with the auxin, and the cello-oligosaccharides increased with stem elongation even after 2 h. The total activity of cellulase did not increase for up to 4 h.
Independent Regulation of Flowering by Phytochrome B and Gibberellins in Arabidopsis1
Authors: Blázquez Miguel A. , Weigel Detlef 
Published: July 2002

Abstract
Phytochromes and gibberellins (GAs) coordinately regulate multiple aspects of Arabidopsis development. Phytochrome B (PHYB) promotes seed germination by increasing GA biosynthesis, but inhibits hypocotyl elongation by decreasing the responsiveness to GAs. Later in the life cycle of the plant, PHYB and GAs have opposite effects on flowering. PHYB delays flowering, while GAs promote flowering, particularly under noninductive photoperiods. To learn how PHYB and GAs interact in the control of flowering, we have analyzed the effect of a phyB mutation on flowering time and on the expression of the floral meristem-identity geneLFY(LEAFY). We show that the early flowering caused by phyBcorrelated with an increase in LFY expression, which complements our previous finding that GAs are required for activation of LFY under noninductive photoperiods (M.A. Blázquez, R. Green, O. Nilsson, M.R. Sussman, D. Weigel [1998] Plant Cell 10: 791−800). Since phyB did not change the GA responsiveness of the LFY promoter and suppressed the lack of flowering of severe GA-deficient mutants under short days, we propose that PHYB modulates flowering time at least partially through a GA-independent pathway. Interestingly, the effects of PHYB on flowering do not seem to be mediated by transcriptional up-regulation of genes such as CO (CONSTANS) andFT (Flowering locusT), which are known to mediate the effects of the photoperiod-dependent floral-induction pathway.
Isolation and Characterization of HvNRT2.3and HvNRT2.4, cDNAs Encoding High-Affinity Nitrate Transporters from Roots of Barley
Authors: Vidmar Joseph John , Zhuo Degen , Siddiqi M. Yaeesh , Glass Anthony D.M. 
Published: July 2002

Abstract
Two full-length cDNAs,HvNRT2.3 and HvNRT2.4, were isolated from roots of barley (Hordeum vulgare), using reverse transcriptase-PCR and RACE-PCR. The corresponding polypeptides, consisting of 507 amino acids (molecular masses of 54.6 kD), belong to the major facilitator superfamily (MFS), and are closely related (&gt;87% identity) to those encoded by HvNRT2.1 andHvNRT2.2 (formerly BCH1 andBCH2, respectively) from roots of barley. The latter are considered to encode inducible high-affinity NO3 − transporters (Trueman et al., 1996).HvNRT2 transcripts were undetectable in NO3 −-deprived plants. Following exposure to either NO3 − or NO2 −, transcript abundance and 13NO3 −influx increased to a maximum by 6 to 12 h, then declined inHvNRT2.1, HvNRT2.2, andHvNRT2.3. The pattern of HvNRT2.4transcript abundance was different, remaining high after achieving peak abundance. When external NO3 − concentrations were varied from 0 to 500 μm under steady-state conditions of NO3 − supply,HvNRT2 transcript accumulation and13NO3 − influx were highest in 50 μm NO3 − -grown plants. When NH4 + was provided together with NO3 −, transcript accumulation during the first 2 h was similar to that due to NO3 − alone, but by 4 h the transcript level was significantly reduced. HvNRT2 transcript was undetectable in leaf tissues.
Purification and Characterization of a Novel Pumpkin Short-Chain Acyl-Coenzyme A Oxidase with Structural Similarity to Acyl-Coenzyme A Dehydrogenases
Authors: De Bellis Luigi , Gonzali Silvia , Alpi Amedeo , Hayashi Hiroshi , Hayashi Makoto , Nishimura Mikio 
Published: July 2002

Abstract
A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs.
Structural Change Caused by Substitution of Nd for Sm in (Nd, Sm)AlO3: Application of Synchrotron High-Resolution Powder X-ray Diffraction
Authors: Saitow Akihiro , Yoshikawa Akira , Horiuchi Hiroyuki , Shishido Toetsu , Fukuda Tsuguo , Tanaka Masahiko , Mori Takeharu , Sasaki Satoshi 
Published: July 2002

Structural change caused by substitution of Nd for Sm in perovskite (Nd, Sm)AlO3was analysed by application of high-resolution powder X-ray diffraction using synchrotron radiation. The parallel, well monochromated and bright incident X-rays improved the full width at half-maximum (FWHM) to 0.027° in a wide 2θ range for the standard Si powder. Applying this high-resolution optical system, the lattice parameters of the solid solution (Ndx, Sm_{1-x})AlO3were precisely analysed for the phases fromx = 0.0 to 1.0 with an interval of 0.2. The lattice parameters of a series ofRAlO3vary systematically with the average ionic radii of R^{3+}, accompanying a structural change from orthorhombic to a trigonal system at around R^{3+}=1.11 Å corresponding to average ionic radii of 0.7{\rm Nd}^{3+}+0.3{\rm Sm}^{3+}. In orthorhombic phases, deformation of the crystal lattice from its ideal cubic lattice is minimized at aroundx = 0.0–0.2 in (Ndx, Sm_{1-x})AlO3and increased with increasing average ionic radii of Nd^{3+} and Sm^{3+}. The structure changes from orthorhombic to trigonal at aroundx = 0.7.
Mechanistic aspects of cyanogenesis from active‐site mutant Ser80Ala of hydroxynitrile lyase from Manihot esculenta in complex with acetone cyanohydrin
Authors: Lauble Hanspeter , Miehlich Burkhard , Förster Siegfried , Wajant Harald , Effenberger Franz 
Published: July 2002

AbstractThe structure and function of hydroxynitrile lyase from Manihot esculenta (MeHNL) have been analyzed by X‐ray crystallography and site‐directed mutagenesis. The crystal structure of the MeHNL–S80A mutant enzyme has been refined to an R‐factor of 18.0% against diffraction data to 2.1‐Å resolution. The three‐dimensional structure of the MeHNL–S80A–acetone cyanohydrin complex was determined at 2.2‐Å resolution and refined to an R‐factor of 18.7%. Thr11 and Cys81 involved in substrate binding have been substituted by Ala in site‐directed mutagenesis. The kinetic measurements of these mutant enzymes are presented. Combined with structural data, the results support a mechanism for cyanogenesis in which His236 as a general base abstracts a proton from Ser80, thereby allowing proton transfer from the hydroxyl group of acetone cyanohydrin to Ser80. The His236 imidazolium cation then facilitates the leaving of the nitrile group by proton donating.
The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase
Authors: Wurth Christine , Kessler Ulrich , Vogt Joachim , Schulz Georg E. , Folkers Gerd , Scapozza Leonardo 
Published: July 2002

AbstractThe structure of Herpes simplex virus type 1 thymidine kinase (TKHSV1) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TKHSV1, measured by thermal denaturation experiments, far‐UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand‐free TKHSV1 is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of TKHSV1 in complex with two new ligands, HPT and HMTT, refined to 2.2 Å are presented. Although TKHSV1:HPT does not exhibit any significant deviations from the model of TKHSV1:dT, the TKHSV1:HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TKHSV1substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.