1,721,104 research outputs found
Effects of Substituent and Scaffold Changes on the Inhibition of Human P5C Reductase by Phenyl-Substituted Aminomethylene Bisphosphonates
No full textBeing overexpressed in many cancer types and related to tumor invasiveness, the activity of P5C reductase represents a promising target for cancer therapy, yet no effective inhibitors have been identified so far. Several phenyl-substituted aminomethylenebisphosphonic acids had been found to inhibit the plant enzyme in the micro- to millimolar range. The two most active compounds were previously shown to be remarkably active against human P5C reductase (PYCR1, gene ID 5831). To investigate their structure–activity relationships, the human enzyme was heterogously expressed in E. coli, affinity purified and assayed in the presence of increasing concentrations of various aminobisphosphonates differing in substituents on the phenyl ring, using either NADH or NADPH as the electron donor. Some analogues, namely aminoethylenebisphosphonates, hydroxybisphosphonates, aminophosphonates and hydroxyphosphonates, were also evaluated. Results allowed to define the chemical features required for effective inhibition. The aminobisphosphonic moiety was found essential for activity, which was enhanced by the presence of electron-withdrawing substituents on the phenyl ring, provided that an optimal steric hindrance is not exceeded. These results could open up new perspectives on the synthesis of effective inhibitors of human P5C reductase to be used in chemotherapy
Differential in vitro responses of rice cultivars to Italian lineages of the blast pathogen Pyricularia grisea. 2. Aromatic biosynthesis.
No full textPhenylalanine ammonia lyase specific activity levels were measured in suspension cultured cells of six rice cultivars following the treatment with cell wall hydrolysates prepared from seven Pyricularia grisea strains. Early after elicitation, even low hydrolysate concentrations were able to induce a significant increase of enzyme levels. However, neither rice genotypes showing differential sensitivity to blast reacted differently, nor elicitors obtained from various pathotypes induced different reactions. At a later stage, higher hydrolysate concentrations were required to trigger maximal enzyme induction. In this case also, only slight variations were detected in suspension cultures of a given cultivar treated with different elicitors. On the contrary, highly significant differences were disclosed among plant genotypes. A remarkable relationship was evident between the mean increase in phenylalanine ammonia lyase activity and the overall resistance to blast at the plant level. This trait could therefore represent a useful tool aiming at the selection for increased blast tolerance
Properties of the 5-enol-pyruvyl-shikimate-3-phosphate synthase isoforms isolated from maize cultured cells
No full textTwo isoforms of the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase, previously purified from maize cultured cells and both found to be functionally located in the plastid while showing different pattern of expression during the culture growth cycle, were characterized with respect to physical and functional properties. A high degree of similarity was found as to their structural features, with the only exception of a slight difference in molecular mass. Both enzyme activities were extremely susceptible to the inhibition brought about by the herbicide glyphosate, and not subjected to feed-back regulation by aromatic amino acids or shikimate pathway intermediates. A more pronounced difference was evident in thermal stability, and catalytic efficiency as judged from the comparison of catalytic constants, affinities for the two substrates and activation energy values. The isozyme detectable in actively-proliferating cells, when the plant cell demand for aromatic amino acids increases, resulted the more stable and efficient. Data are consistent with the hypothesis of an isoform-based mechanism of enzyme level modulation in plant aromatic metabolism
Differential expression of 5-enol-pyruvyl-shikimate-3-phosphate synthase isoforms in elicitor-treated, cultured maize cells
No full textThe expression of two 5-enol-pyruvyl-shikimate-3-phosphate synthase (EC 2.5.1.19) isoforms was investigated in Zea mays L. suspension-cultured cells following exposure to a fungal elicitor. Activity levels of isozyme II specifically increased soon after treatment, in strict connection with induction of phenylalanine ammonia-lyase (PAL) and attainment of a new free-phenylalanine homeostasis at a higher concentration. However, a few days later, activity of the other enzyme form was also significantly enhanced, concomitant with a sharp rise in overall amino acid content, a further increase in PAL level and a resumption of cell lysis. Besides strengthening the hypothesis that an entire set of genes encoding for shikimate pathway enzymes (whose expression is specifically involved in plant dynamic defence) may exist, a general change in the levels of several amino acids seems to point towards a reprogramming of their metabolism in elicited cells
Purification and properties of a pyruvate carboligase from Zea mays cultured cellsfn1fn1Part 2 in the series Acetoin synthesis in higher plants.
No full textAn enzyme able to catalyze the synthesis of acetoin (3-hydroxy-2-butanon) from either pyruvate or acetaldehyde was isolated, partially purified and characterized from maize (Zea mays L. cv Black Mexican Sweet) cultured cells. It exhibited a maximal rate at neutral pH values, and strictly required thiamine pyrophosphate and a divalent cation for activity; on the contrary, unlike bacterial pyruvate oxidases, flavin was not required. Apparent Michaelis constants were 260 +/- 20 micromolar for pyruvate and 24 +/- 7 micromolar for acetaldehyde. Both substrate affinity and specificity were notably higher than those of pyruvate decarboxylase, an enzyme that also synthesizes acetoin as by-product. The partially purified protein was unable to catalyze the formation of other possible products of pyruvate decarboxylation, thus carboligase appears to be its main activity. Results suggest that acetoin synthesis may be of physiological significance in plants
Purification and properties of a cytosolic glutamine synthetase expressed in Nicotiana plumbaginifolia cultured cells
No full textGlutamine synthetase (EC 6.3.1.2) was purified and characterized from leaf protoplast-derived suspension cultured cells of Nicotiana plumbaginifolia. Maximal specific activity observed reflected a purification of over 1 500-fold, with a yield of about 40%. The native protein appeared to be an octamer, with subunits of a molecular mass of 37 kDa. Subcellular fractionation experiments accounted for a cytosolic localization of the enzyme. No evidence for multiple enzyme forms was found following either anion-exchange chromatography or native polyacrylamide gel electrophoresis. Results also suggest that in the absence of intercellular transport, type-1 glutamine synthetase may function preferentially to assimilate ammonia from primary nitrate reduction
Osmotic adjustments in a psychrophilic alga, Xanthonema sp. (Xanthophyceae)
No full textExtremophile microalgae show a remarkable ability to cope with harsh environments, yet still relatively little is known about the molecular basis for such tolerance. In this work the susceptibility of a psychrophylic alga isolated from alpine snowbanks, Xanthonema sp., to either water or salt stress conditions was assessed, and mechanisms for osmotic adjustment were investigated. Cultures were treated with increasing concentrations of either salts or non-permeant solutes, as polyethylen glycol, and the resulting effect on growth rate was measured. Both the accumulation of compatible osmolytes and the activity of cation transporters were studied in response to the exposure to hyperosmotic conditions. Xanthonema showed a differential sensitivity to osmotic and ionic stress, with a noteworthy tolerance to NaCl. No evidence was found supporting an osmo-induced intracellular accumulation of the most common osmoprotectants. Salt tolerance seems to rely upon the inducible expression of an amiloride-resistant Na+/H+ antiporter. Since in snow fields osmotic unbalance due to freeze/thaw is more likely to occur than excess salts, results suggest an allochthonous origin of the strain
Glyphosate tolerance in maize (Zea mays L.) 1. Differential response among inbred lines.
No full textVariation in susceptibility to the safe broad-spectrum herbicide glyphosate was investigated in maize. Eleven inbred lines, grown in a growth chamber, were evaluated for their tolerance to the herbicide at 2.4 mM (0.2 kg a.i. in 400 I ha-1 of water). Following treatment with glyphosate at the three-leaf stage, significant variation in damage, expressed as visual injury ratings scored 7, 14 and 21 days after the application of the herbicide, was found. Effects on dry weight and shoot height were consistent with visual scores and the carbon-exchange rate was found to be a sensitive index of differential injury.
Biochemical characterization of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase, the main target of the herbicide, ruled out the possibility that this differential susceptibility was due to variations in the sensitivity of the enzyme. On the contrary, a positive correlation was found between in vivo tolerance and EPSP synthase levels, measured at different stages during seedling growth. This result suggests that a naturally occurring difference in EPSP synthase levels in the tissues may contribute to the differential response observed in vivo in maize inbreds
Herbicidally active aminomethylenebisphosphonic acids
No full textHerbicidal derivatives of aminomethylenebisphosphonic acids discovered over 20 years ago were considered to be some kind of curiosity until 1995. Renewed interest in these compounds brought controversies about their modes and cellular targets of action
5-Enol-pyruvyl-shikimate-3-phosphate synthase from Zea mays cultured cells: purification and properties
No full textThe shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays L. var Black Mexican Sweet) cells. Homogeneous enzyme preparations were obtained by a four-step procedure using ammonium sulfate fractionation, anion- and cation-exchange chromatography, and substrate elution from a cellulose phosphate column. The last step resulted in two well-separated activities of about the same molecular weight. A 2000- to 3000-fold purification, with an overall recovery of one-fourth of the initial activity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, activation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. Two EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. This allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throughout, whereas the other increased in exponentially growing cells and declined in late-logarithmic phase. The analysis of highly purified plastid preparations demonstrated a plastidial localization of both proteins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on defense-related aromatic biosynthesis in higher plants, are discussed
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