RESUMO
Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG beta-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of beta-subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of beta-galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1-->4)beta-D-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably beta-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.
Assuntos
Parede Celular/metabolismo , Frutas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Parede Celular/genética , Enzimas/genética , Enzimas/metabolismo , Frutas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Polissacarídeos/metabolismoRESUMO
A lipophilicity constrained library of 5-carboxamido 1-benzyl-3-(3-dimethylaminopropyloxy)-1H-pyrazoles was prepared by solution-phase parallel synthesis with removal of acidic by-products using the strongly basic MP-carbonate resin. Compounds show both activation of soluble guanylate cyclase and inhibition of platelet aggregation. Compound 12 also shows 22% oral bioavailability in rats.
Assuntos
Ativadores de Enzimas/síntese química , Ativadores de Enzimas/farmacologia , Guanilato Ciclase/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Administração Oral , Animais , Disponibilidade Biológica , Colágeno/farmacologia , GMP Cíclico/análise , Concentração Inibidora 50 , Pirazóis/síntese química , Pirazóis/farmacologia , RatosRESUMO
Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.
Assuntos
Guanilato Ciclase/metabolismo , Indazóis/síntese química , Óxido Nítrico/metabolismo , Pirazóis/síntese química , Animais , Ativação Enzimática , Humanos , Técnicas In Vitro , Indazóis/química , Indazóis/farmacocinética , Indazóis/farmacologia , Masculino , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacocinética , Inibidores da Agregação Plaquetária/farmacologia , Pirazóis/química , Pirazóis/farmacocinética , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Solubilidade , Relação Estrutura-AtividadeRESUMO
Plants of tomato (Lycopersicon esculentum Mill. cv. T5) were transformed with an antisense endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) Cel2 transgene under the control of the constitutive cauliflower mosaic virus 35S promoter in order to suppress mRNA accumulation of Cel2. In two independent transgenic lines, Cel2 mRNA abundance was reduced by >95% in ripe fruit pericarp and ca. 80% in fruit abscission zones relative to non-transgenic controls. In both transgenic lines the softening of antisense Cel2 fruit pericarp measured using stress-relaxation analysis was indistinguishable from control fruit. No differences in ethylene evolution were observed between fruit of control and antisense Cel2 genotypes. However, in fruit abscission zones the suppression of Cel2 mRNA accumulation caused a significant (P<0.001) increase in the force required to cause breakage of the abscission zone at 4 days post breaker, an increase of 27% in one transgenic line and of 46% in the other transgenic line. Thus the Cel2 gene product contributes to cell wall disassembly occurring in cell separation during fruit abscission, but its role, if any, in softening or textural changes occurring in fruit pericarp during ripening was not revealed by suppression of Cel2 gene expression.
Assuntos
Celulase/genética , RNA Antissenso/genética , RNA Mensageiro/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Etilenos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genótipo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genéticaRESUMO
cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955-5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.
Assuntos
Proteínas de Plantas/genética , Solanum lycopersicum/genética , Sequência de Aminoácidos , DNA Complementar/química , DNA de Plantas/química , Frutas/fisiologia , Solanum lycopersicum/fisiologia , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/fisiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Alinhamento de SequênciaRESUMO
A cDNA (Cel1) encoding an endo-1,4-beta-glucanase (EGase) was isolated from ripe fruit of strawberry (Fragaria x ananassa). The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one potential N-glycosylation site. Southern- blot analysis of genomic DNA from F. x ananassa, an octoploid species, and that from the diploid species Fragaria vesca indicated that the Cel1 gene is a member of a divergent multigene family. In fruit, Cel1 mRNA was first detected at the white stage of development, and at the onset of ripening, coincident with anthocyanin accumulation, Cel1 mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent. Antibodies raised to Cel1 protein detected a protein of 62 kD only in ripening fruit. Upon deachenation of young white fruit to remove the source of endogenous auxins, ripening, as visualized by anthocyanin accumulation, and Cel1 mRNA accumulation were both accelerated. Conversely, auxin treatment of white fruit repressed accumulation of both Cel1 mRNA and ripening. These results indicate that strawberry Cel1 is a ripening-specific and auxin-repressed EGase, which is regulated during ripening by a decline in auxin levels originating from the achenes.
Assuntos
Celulase/genética , Frutas/enzimologia , Frutas/genética , Genes de Plantas , Sequência de Aminoácidos , Celulose 1,4-beta-Celobiosidase , DNA Complementar/genética , DNA de Plantas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.
Assuntos
Membrana Celular/enzimologia , Celulase/isolamento & purificação , Complexo de Golgi/enzimologia , Proteínas de Membrana/isolamento & purificação , Solanum lycopersicum/genética , Sequência de Aminoácidos , Biomarcadores , Compartimento Celular , Fracionamento Celular , Celulase/genética , DNA Complementar/genética , Biblioteca Gênica , Hipocótilo/enzimologia , Solanum lycopersicum/enzimologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , Reguladores de Crescimento de Plantas/farmacologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição TecidualRESUMO
Plant developmental processes involving modifications to cell wall structure, such as cell expansion, organ abscission and fruit ripening, are accompanied by increased enzyme activity and mRNA abundance of endo-1,4-beta-glucanases (EGases). An EGase cDNA clone, Cel4, isolated from tomato (Lycopersicon esculentum) has been shown to be identical to a tomato pistil-predominant EGase cDNA, TPP18. In addition to its previously reported expression during certain stages of early pistil development, Cel4 mRNA was also detected at high levels in the growing zones of etiolated hypocotyls (about 2.5-fold less than in pistils) and in young expanding leaves (about 3.5-fold less than in pistils). The abundance of Cel4 mRNA declined precipitously in older tissues as cells became fully expanded, and was barely detectable in mature vegetative tissues. Cel4 mRNA abundance was also low in abscission zones, and did not increase as abscission progressed. In fruit, Cel4 mRNA was present at low levels during fruit expansion, but was essentially absent during subsequent fruit development and ripening. Treatment of etiolated hypocotyls with ethylene or high concentrations of auxin sufficient to induce rapid lateral cell expansion and hypocotyl swelling also brought about an approximate doubling of Cel4 mRNA abundance, suggesting that Cel4 mRNA accumulation may be promoted directly or indirectly by ethylene. Thus, accumulation of Cel4 mRNA was found to be correlated with rapid cell expansion in pistils, hypocotyls and leaves.
Assuntos
Celulase/biossíntese , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/enzimologia , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Solanum lycopersicum/crescimento & desenvolvimento , RNA Mensageiro/biossíntese , Transcrição Gênica , Verduras/enzimologia , Verduras/genéticaRESUMO
Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.
RESUMO
The mRNA accumulation of two endo-1,4-[beta]-D-glucanase genes, Cel1 and Cel2, was examined in the pericarp and locules throughout the development of normal tomato (Lycopersicon esculentum) fruit and the ripening-impaired mutants rin and Nr. Both Cel1 and Cel2 were expressed transiently at the earliest stages of fruit development during a period corresponding to cell division and early cell expansion. In the pericarp, the mRNA abundance of both genes increased markedly at the breaker stage; the level of Cel1 mRNA decreased later in ripening, and that of Cel2 increased progressively. Cel2 mRNA levels also increased at the breaker stage in locules but after initial locule liquefaction was already complete. In rin fruit mRNA abundance of Cel1 was reduced and Cel2 was virtually absent, whereas in Nr Cel1 was expressed at wild-type levels and Cel2 was reduced. In wild-type fruit ethylene treatment slightly promoted the mRNA accumulation of both genes. In rin fruit ethylene treatment strongly increased the mRNA abundance of Cel1 to an extent greater than in wild-type fruit, but Cel2 mRNA was absent even after ethylene treatment. These two endo-1,4-[beta]-D-glucanase genes, therefore, do not show coordinated expression during fruit development and are subject to distinct regulatory control. These results suggest that the product of the Cel2 gene contributes to ripening-associated cell-wall changes.
RESUMO
A cDNA encoding a xyloglucan endotransglycosylase (XET) homolog was isolated from nasturtium (Tropaeolum majus) epicotyl RNA. The deduced protein encoded by the corresponding gene, termed XET1, was substantially divergent from a previously isolated nasturtium XET (NXG1) expressed in germinating seed cotyledons but was highly homologous to XET genes isolated from vegetative tissues of several distantly related species. XET1 was expressed at the level of mRNA accumulation in all vegetative tissues examined (root, epicotyl, stem, and leaf) except in germinating cotyledons. Conversely, NXG1 exhibited the opposite pattern of expression and its mRNA was detected exclusively in cotyledons. Both XET1 and NXG1 were apparently encoded by single genes. Protein extracts from epicotyls or germinating cotyledons, in which XET1 or NXG1 are specifically expressed respectively, exhibited XET activity when assayed using two different xyloglucan substrates. XET activity from epicotyl extracts used nonfucosylated seed amyloid xyloglucan or fucosylated stem xyloglucan as a substrate with equal facility, whereas XET activity from cotyledon extracts had a significantly higher activity against nonfucosylated xyloglucan. The existence in a single species of two XETs possessing divergent amino acid sequences, mutually exclusive patterns of expression, and potentially different activities against xyloglucan substrates demonstrates the existence of different classes of XET and suggests differing roles in vivo.
Assuntos
Expressão Gênica , Glicosiltransferases/biossíntese , Isoenzimas/biossíntese , Filogenia , Plantas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , Primers do DNA , DNA Complementar , Genes de Plantas , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Cinética , Dados de Sequência Molecular , Especificidade de Órgãos , Plantas/genética , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization.
Assuntos
Expressão Gênica , NADH NADPH Oxirredutases/biossíntese , Solanum lycopersicum/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , DNA Complementar , Indução Enzimática , Biblioteca Gênica , Solanum lycopersicum/enzimologia , Dados de Sequência Molecular , NADH NADPH Oxirredutases/metabolismo , Folhas de Planta , Raízes de Plantas , Caules de Planta , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Ferimentos e LesõesRESUMO
We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti-carbohydrate Fabs, one specific for a Brucella cell-surface polysaccharide and the second for the human blood group A determinant. Very similar VL amino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized VH genes were fused to a previously made sequence encoding the C(gamma 1)1 domain as an alternative to synthesis of the natural C gamma 2b 1 and C mu 1 sequences. The VL genes were initially coupled to a synthetic C kappa gene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the C kappa domains with a C lambda 1 domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The C kappa and C lambda 1 forms of each of the Brucella and blood group A Fabs, with His5 fusions at the C-termini of the Fd chains, were purified by immobilized metal affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Carboidratos/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Sequência de Bases , Brucella , Clonagem Molecular , Escherichia coli , Código Genético , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Dados de Sequência MolecularRESUMO
The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.
Assuntos
Sítios de Ligação de Anticorpos , Carboidratos/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Sequência de Aminoácidos , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/genética , Sequência de Bases , Sítios de Ligação de Anticorpos/genética , Western Blotting , Calorimetria , Metabolismo dos Carboidratos , Sequência de Carboidratos , DNA Bacteriano , Ligação de Hidrogênio , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Salmonella/imunologiaRESUMO
GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.
Assuntos
Fucosiltransferases/isolamento & purificação , Plantas/enzimologia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Fucosiltransferases/química , Fucosiltransferases/metabolismo , Focalização Isoelétrica , Cinética , Peso Molecular , SolubilidadeRESUMO
The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.
Assuntos
Polissacarídeos Bacterianos/genética , Salmonella/genética , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo , Sequência de Bases , Sítios de Ligação de Anticorpos , Técnicas Imunoenzimáticas , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Antígenos O , Oligodesoxirribonucleotídeos , Polissacarídeos Bacterianos/imunologiaRESUMO
Rapid mobilisation of storage products, including xyloglucan, in cotyledons of germinating nasturtium (Tropaeolum majus L.) normally starts about 7-8 d after imbibition and growth of the seedling at 20-25° C. Levels of activity of endo-1,4-ß-glucanase (EC 3.2.1.4) in cotyledons, as assayed viscometrically with xyloglucan as substrate, varied in parallel with the rate of breakdown of xyloglucan. When cotyledons were excised from the seedling axis and incubated on moist filter paper at any point before 7 d, the catabolic reactions which normally occurred in the intact seedling were suspended. If, however, cotyledons excised at 8 d were incubated in 10(-6) M 2,4-dichlorophenoxyacetic acid, a rise in endo-1,4-ß-glucanase (xyloglucanase) activity was observed and a sharp decrease in fresh and dry weight as well as xyloglucan levels ensued at rates comparable to those observed in cotyledons attached to the seedling. Neither gibberellin nor kinetin treatments promoted xyloglucan breakdown or enhanced xyloglucanase activity. Addition of auxin to excised cotyledons before 7 d did not evoke premature breakdown, indicating that the tissue became receptive to auxin only at this time. The triggering process took place in darkness and was unaffected by various light-dark cycles. It is concluded that the sudden degradation of xyloglucan which occurs in nasturtium seeds about a week after germination begins is the result of enhanced activity of a depolymerizing xyloglucanase, this activity being evoked by auxin originating in the emerging seedling axis.
RESUMO
Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-[(14)C] fucose and UDP-[(14)C]xylose to xyloglucan occurred mainly in dictyosomeenriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with [(3)H]fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.
RESUMO
Microsomal fractions from wheat coleoptiles and pea stems contain a microsomal ATPase activity that requires divalent cations (Ca2+ is more effective than Mg2+) and shows further stimulation by KCl. The effects of added indoleacetic acid were inconclusive. Cytochemical studies on both species showed most pronounced staining for ATPase in the plasmalemma at pH 7.0. However, at pH 5.5, the coleoptile cells showed heaviest staining for ATPase in the endoplasmic reticulum and dictyosomes. The results are discussed with regard to the postulated role of ATPase activity in relation to proton pumping and plant cell elongation.
Assuntos
Adenosina Trifosfatases/análise , Fabaceae/enzimologia , Microssomos/análise , Plantas Medicinais , Triticum/enzimologia , Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Ácidos Indolacéticos/farmacologia , Canais Iônicos/metabolismo , Magnésio/metabolismoRESUMO
The effects of peeling and wounding on the indole-3-acetic acid (IAA) and fusicoccin (FC) growth response of etiolated Pisum sativum L. cv. Alaska stem tissue were examined. Over a 5 h growth period, peeling was found to virtually eliminate the IAA response, but about 30% of the FC response remained. In contrast, unpeeled segments wounded with six vertical slits exhibited significant responses to both IAA and FC, indicating that peeling does not act by damaging the tissue. Microscopy showed that the epidermis was removed intact and that the underlying tissue was essentially undamaged. Neither the addition of 2% sucrose to the incubation medium nor the use of a range of IAA concentrations down to 10(-8) M restored IAA-induced growth in peeled segments, suggesting that lack of osmotic solutes and supra-optimal uptake of IAA were not important factors over this time period. It is concluded that, although the possibility remains that peeling merely allows leakage of hydrogen ions into the medium, it seems more likely that peeling off the epidermis removes the auxin responsive tissue.