RESUMEN
We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.
Asunto(s)
Proteínas Anticongelantes/química , Lolium/metabolismo , Péptidos/química , Animales , Proteínas Anticongelantes/metabolismo , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Peces , Calor , Hielo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , TemperaturaRESUMEN
Galactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP-mannose dependent (1-->4)-beta-D-mannan synthase and a UDP-galactose dependent (1-->6)-alpha-D-galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta-(1-->4)-linked D-manno-oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactose-substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan alpha-galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane-spanning helix, was expressed.
Asunto(s)
Pared Celular/enzimología , Fabaceae/enzimología , Galactosiltransferasas/aislamiento & purificación , Mananos/biosíntesis , Plantas Medicinales , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Galactosa/análogos & derivados , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Semillas/enzimología , Análisis de Secuencia de ADN , Análisis de Secuencia de ProteínaRESUMEN
Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Isoenzimas/metabolismo , Solanum tuberosum/enzimología , Almidón/química , Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Secuencia de Aminoácidos , Secuencia de Bases , Conformación de Carbohidratos , Cartilla de ADN , ADN Complementario , Escherichia coli/genética , Isoenzimas/química , Isoenzimas/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Homología de Secuencia de AminoácidoRESUMEN
Many organisms adapted to live at subzero temperatures express antifreeze proteins that improve their tolerance to freezing. Although structurally diverse, all antifreeze proteins interact with ice surfaces, depress the freezing temperature of aqueous solutions, and inhibit ice crystal growth. A protein purified from carrot shares these functional features with antifreeze proteins of fish. Expression of the carrot complementary DNA in tobacco resulted in the accumulation of antifreeze activity in the apoplast of plants grown at greenhouse temperatures. The sequence of carrot antifreeze protein is similar to that of polygalacturonase inhibitor proteins and contains leucine-rich repeats.
Asunto(s)
Daucus carota/química , Glicoproteínas/química , Glicoproteínas/fisiología , Hielo , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Secuencia de Aminoácidos , Proteínas Anticongelantes , Clonación Molecular , Cristalización , ADN Complementario , Daucus carota/fisiología , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Glicosilación , Punto Isoeléctrico , Leucina/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas/química , Plantas Modificadas Genéticamente , Plantas Tóxicas , NicotianaRESUMEN
Potato (Solanum tuberosum) tuber lectin is a chitin-binding, hydroxyproline-rich glycoprotein, which may be involved in the defence mechanism of the plant. We had previously obtained evidence that it consists of at least two very dissimilar domains. The aim was to use a combination of accurate determinations of molecular weight and protein sequencing to gain more accurate information on the domains. Accurate determinations of the molecular weight of the lectin by a MALDI mass spectrometer have shown that the subunit molecular weight is 65,500 (+/- 1100) and that of a totally deglycosylated sample is 31,250 (+/- 30). This means that the lectin is 52.3 (+/- 1)% carbohydrate with a considerable number of glycoforms being present. Partial sequences and other analyses are consistent with the existence of three distinct domains. These are: (1) an N-terminal region which is rich in proline but poor in hydroxyproline; (2) a glycosylated region with a glycosylated molecular weight of 45,300 (+/- 1100) and a deglycosylated molecular weight of 11,050 (+/- 50) which is extremely rich in glycosylated hydroxyproline residues with a similar sequence to extensins; and (3) a cystine-rich domain which has the sugar binding site shows partial conservation of a repeated motif common to many chitin-binding proteins of the hevin family including wheat-germ agglutinin. The closest similarity seems to be to the sequence of potato basic chitinase.
Asunto(s)
Lectinas/química , Lectinas/genética , Solanum tuberosum/química , Solanum tuberosum/genética , Aglutininas del Germen de Trigo/genética , Secuencia de Aminoácidos , Sitios de Unión , Quitina/metabolismo , Secuencia Conservada , Cistina/química , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hidroxiprolina/química , Lectinas/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Peso Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Lectinas de Plantas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , Solanum tuberosum/metabolismoRESUMEN
In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.
Asunto(s)
Isoenzimas/metabolismo , Pisum sativum/enzimología , Almidón Sintasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Escherichia coli , Isoenzimas/biosíntesis , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Semillas/enzimología , Homología de Secuencia de Aminoácido , Solanum tuberosum , Almidón , Almidón Sintasa/biosíntesis , Almidón Sintasa/aislamiento & purificaciónRESUMEN
The major isoform of starch synthase from the soluble fraction of developing potato tubers has been purified and used to prepare an antibody and isolate a cDNA. The protein is 140 kD, and it is distinctly different in predicted primary amino acid sequence from other isoforms of the enzyme thus far described. Immunoinhibition and immunoblotting experiments and analysis of tubers in which activity of the isoform was reduced through expression of antisense mRNA revealed that the isoform accounts for approximately 80% of the activity in the soluble fraction of the tuber and that it is also bound to starch granules. Severe reductions in activity had no discernible effect on starch content or amylose-to-amylopectin ratio of starch in tubers. However, they caused a profound change in the morphology of starch granules, indicative of important underlying changes in the structure of starch polymers within the granule.
Asunto(s)
Solanum tuberosum/enzimología , Almidón Sintasa/química , Almidón Sintasa/metabolismo , Secuencia de Aminoácidos , Anticuerpos , Bacterias/enzimología , Cromatografía por Intercambio Iónico , Clonación Molecular , Citosol/enzimología , ADN Complementario , Expresión Génica , Cinética , Datos de Secuencia Molecular , Filogenia , Raíces de Plantas , ARN sin Sentido , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Almidón Sintasa/aislamiento & purificaciónRESUMEN
An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity. The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.
Asunto(s)
Isoenzimas/metabolismo , Tallos de la Planta/enzimología , Solanum tuberosum/enzimología , Almidón Sintasa/metabolismo , Almidón/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN Complementario/genética , Escherichia coli/genética , Immunoblotting , Isoenzimas/biosíntesis , Isoenzimas/genética , Datos de Secuencia Molecular , Orgánulos/enzimología , Tallos de la Planta/genética , Pruebas de Precipitina , ARN sin Sentido , Proteínas Recombinantes/biosíntesis , Solanum tuberosum/genética , Almidón Sintasa/biosíntesis , Almidón Sintasa/genética , Fracciones Subcelulares/enzimología , Transformación GenéticaRESUMEN
Characterization of Pisum (pea) seed trypsin inhibitors (TI) and their corresponding cDNAs indicates that the pea TI gene family contains two genes. The existence of multiple TI isoforms can be attributed to post-translational modifications of primary gene products. Post-translational processing at the C-terminus during the desiccation stage of seed development results in the appearance of TI isoforms with increased affinity for the target enzyme, trypsin.
Asunto(s)
Pisum sativum/genética , Inhibidores de Tripsina/genética , Secuencia de Aminoácidos , Cromatografía de Afinidad , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Genes de Plantas , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Pisum sativum/química , Pisum sativum/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Inhibidores de Tripsina/aislamiento & purificación , Inhibidores de Tripsina/metabolismoRESUMEN
In mammals, an AMP-activated protein kinase (AMPK) phosphorylates both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro and has been proposed to play a major role in the regulation of lipid metabolism in vivo. We report here the primary sequence of rat AMPK and show that antibodies raised against synthetic peptides based on the deduced sequence of AMPK immunoprecipitate AMPK activity from rat liver extracts. AMPK has a remarkable degree of sequence identity to the proteins encoded by the yeast SNF1 gene and the plant RKIN1 gene. SNF1 protein kinase activity is essential for release of genes from glucose repression in Saccharomyces cerevisiae. Expression of cRKIN1 in yeast snf1 mutants restores SNF1 function. These results indicate that AMPK, SNF1, and RKIN1 form part of a family of protein kinases that have been highly conserved throughout evolution. Our results suggest that AMPK may be involved in the regulation of a wide range of metabolic pathways.
Asunto(s)
Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Plantas/enzimología , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/enzimología , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , ADN Complementario/metabolismo , Genes Fúngicos , Genes de Plantas , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hígado/enzimología , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Fosforilación , Plantas/genética , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/metabolismo , ARN Mensajero/biosíntesis , Ratas , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Developing wild-type pea embryos contain two major isoforms of starch synthase and two isoforms of starch-branching enzyme. One of the starch synthases and both starch-branching enzymes occur both in the soluble fraction and tightly bound to starch granules. The other starch synthase, which is very similar to the waxy proteins of other species, is exclusively granule-bound., It is inactive when solubilized in a native form from starch granules, but activity is recovered when the SDS-denatured protein is reconstituted from polyacrylamide gels. Evidence is presented which indicates that all of these proteins become incorporated within the structure of the granule as it grows. It is proposed that the granule-bound waxy protein is active in vivo at the granule surface, whereas the remaining proteins are active in the soluble fraction of the amyloplast. The proteins become trapped within the granule matrix as the polymers they synthesize crystallize around them, and they probably play no further part in polymer synthesis.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Fabaceae/enzimología , Isoenzimas/metabolismo , Plantas Medicinales , Almidón Sintasa/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Secuencia de Aminoácidos , Fabaceae/embriología , Fabaceae/genética , Inmunohistoquímica , Isoenzimas/genética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Solubilidad , Almidón Sintasa/genéticaRESUMEN
A novel xyloglucan-specific endo-(1-->4)-beta-D-glucanase, involved in the post-germinative mobilization of xyloglucan storage reserves, has previously been isolated from nasturtium (Tropaeolum majus L.) seed. Its mode of action has been shown, in vitro, to be one of transglycosylation except at low substrate (glycosylacceptor) concentrations when hydrolysis predominates. Here it is shown that this nasturtium seed xyloglucan endo-transglycosylase is encoded by a single gene which is transcribed and processed to a 1.5 kb mRNA. The isolation and DNA sequence analysis of a cDNA copy of the nasturtium xyloglucan endo-transglycosylase transcript is described. The cDNA encodes a 33.5 kDa precursor polypeptide which is subsequently processed to a 31 kDa mature protein. The precursor incorporates an N-terminal signal sequence which probably contains information relevant to the targeting of the enzyme to the cell wall. The computer-predicted isoelectric point (5.14) and low (approximately 0%) alpha-helix content of the deduced mature protein are in excellent agreement with the experimental data obtained using the purified enzyme. The deduced protein sequence lacks homology with known plant endo-(1-->4)-beta-D-glucanases, consistent with the unique properties of the enzyme. Database searches have revealed that a Brassica protein (meri-5) of previously unknown function, but abundantly expressed in expanding tissue, shares structural identity with the nasturtium xyloglucan endo-transglycosylase. The expression of a xyloglucan endo-transglycosylase in expanding tissue would be consistent with the contention that enzymes of this type are involved in cell wall loosening.
Asunto(s)
Glicosiltransferasas/genética , Plantas/enzimología , Secuencia de Aminoácidos , Aminoácidos/análisis , Secuencia de Bases , Dicroismo Circular , ADN , Precursores Enzimáticos/aislamiento & purificación , Expresión Génica , Genes de Plantas , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Pruebas de Precipitina , ARN Mensajero/metabolismo , Semillas/enzimología , Homología de Secuencia de AminoácidoRESUMEN
The sequence of the 3' 1462nts of RNA-1 of a UK isolate of the fungal-transmitted virus barley mild mosaic (BaMMV) has been determined. An open reading frame encoding the coat protein gene was identified within this region using amino acid sequence information obtained by cyanogen bromide cleavage of virus particles. The amino acid sequence of the full-length coat protein was deduced from the nucleotide sequence. Amino acid sequence comparisons revealed highest homology to the coat protein of barley yellow mosaic virus. In addition, a significant, but limited, number of the amino acid residues that are conserved between aphid-transmitted potyviruses were also conserved between BaMMV and potyviruses.
Asunto(s)
Virus del Mosaico/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cápside/genética , Clonación Molecular , Genes Virales , Hordeum/microbiología , Datos de Secuencia Molecular , Virus del Mosaico/clasificación , ARN Viral/genética , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Tubers of potato (Solanum tuberosum L.) contain a number of chitin-binding proteins which have possible functions in defence against pathogens. A major protein of the tuber is the chitin-binding lectin which has been further characterized with respect to its antigenicity and N-terminal amino acid sequence. By using an antiserum monospecific for tuber lectin in unwounded potato the protein was found in the cytoplasm and vacuole, unusually for a hydroxyproline-rich glycoprotein, but consistent with its soluble nature in subcellular extracts. Little increased synthesis of the lectin precursor or the post-translationally modified form could be demonstrated in excised potato tuber discs. However, after wounding there is increased synthesis of another hydroxyproline-containing glycoprotein of Mr 57,000, which binds to chitin and shares common epitopes with the lectin. In comparison with the tuber lectin, this novel glycoprotein contains less hydroxyproline, but from its overall composition it is clearly not an underhydroxylated form of the tuber lectin. It differed in its N-terminal amino acid sequence and was much less glycosylated, although arabinose was still present. Synthesis of the Mr-57,000 polypeptide began after the initial burst of protein synthesis and increased, reaching a peak at 24 h after wounding. The protein was produced with its enzymes of post-translational modification, prolyl hydroxylase and arabinosyltransferase, concomitantly with the marker enzymes for wounding, phenylalanine ammonia-lyase and membrane-bound phenol oxidase and peroxidase.
Asunto(s)
Quitina/metabolismo , Glicoproteínas/análisis , Lectinas/análisis , Proteínas de Plantas/análisis , Solanum tuberosum/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Western Blotting , Carbohidratos/análisis , Citoplasma/química , Glicoproteínas/química , Glicoproteínas/metabolismo , Hidroxiprolina/análisis , Inmunohistoquímica , Técnicas de Inmunoadsorción , Lectinas/química , Lectinas/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Peso Molecular , Lectinas de Plantas , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Solanum tuberosum/fisiología , Solanum tuberosum/ultraestructura , Vacuolas/químicaRESUMEN
3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.
Asunto(s)
Oxidorreductasas de Alcohol/química , Brassica/enzimología , Ácido Graso Sintasas/química , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Catálisis , Electroforesis en Gel de Poliacrilamida , Ácido Graso Sintasas/metabolismo , Inmunohistoquímica , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Alineación de SecuenciaRESUMEN
cDNA clones encoding the fatty-acid- biosynthetic enzyme NADPH-linked 3-oxoacyl-(acyl carrier protein) (ACP) reductase were isolated from a Brassica napus (rape) developing seed library and from an Arabidopsis thaliana (thale cress) leaf library. The N-terminal end of the coding region shows features typical of a stromal-targeting plastid-transit peptide. The deduced amino acid sequences have 41% and 55% identity respectively with the nodG-gene product of Rhizobium meliloti, one of the host-specific genes that restrict infectivity of this bacterium to a small range of host plants. The probability that the nodG-gene product is a oxoreductase strengthens the hypothesis that some of the host-specific nod-gene products are enzymes which synthesize polyketides that uniquely modify the Rhizobium nodulation signal molecule.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Brassica/genética , Genes Bacterianos , Plantas/genética , Sinorhizobium meliloti/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Oxidorreductasas de Alcohol/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Brassica/enzimología , Clonación Molecular/métodos , Biblioteca de Genes , Datos de Secuencia Molecular , Mapeo Peptídico , Plantas/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
A major wall protein of suspension-cultured cells of French bean has been isolated and characterised. It can be prepared from walls or the culture filtrate and in composition it is particularly rich in proline, valine and glutamic acid/glutamine and contains appreciable amounts of hydroxyproline. The N-terminus shows some glycosylation, while following chemical deglycosylation the first 38 residues were found to be identical to those of proline-rich proteins from soybean. However, the composition of the highly purified Mr-42000 bean protein differs considerably from the soybean proteins and must contain its own specific domains. An antibody was raised and used to demonstrate the inducibility of the Mr-42000 bean protein in response to elicitor action. The protein was found to be mainly localised in the intercellular spaces of the cortical cells of bean hypocotyls and at the wall-plasmalemma interface of xylem vessels, another potentially accessible compartment for pathogens. Following wounding, the protein was found to be generally distributed in the wall of epidermal and cortical cells of the hypocotyls. The Mr-42000 protein is cross reactive with antibodies raised to glycoproteins of the Rhizobium infection thread and the chitin-binding hydroxyproline-rich glycoprotein, potato lectin. These common epitopes together with the previously demonstrated chitin-binding properties of the bean protein indicate a role in host-microbial interactions. Furthermore, the Mr-42000 protein itself bound to the growing hyphal tips of the bean pathogen, Colletotrichum lindemuthianum.
RESUMEN
We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and II (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pI. Thus, lipases I and II have native molecular masses of 50.1 kDa and 55.5 kDa, and pI of 4.61 and 4.47, respectively. Lipases A and B are very similar to lipases I and II with native molecular masses of 53.7 kDa and 48.9 kDa, and pI of 4.71 and 4.50, respectively. Treatment with endo-beta-N-acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.