An unusual case of 'uncompetitive activation' by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings.
Myrosinase (thioglucoside glucohydrolase; EC 22.214.171.124) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sativus (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30% (v/v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gel-filtration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS/PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V(max) and K(m) values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 micromol/min per mg of protein and 23 microM in the absence of ascorbate and 280 micromol/min per mg of protein and 250 microM in the presence of 500 microM ascorbate, respectively. As the ascorbate concentration was increased from 50 to 500 microM, the V(max) and K(m) values increased in parallel, and thus the V(max)/K(m) ratio remained constant. Similarly, raising the concentrations of sinigrin increased the concentration of ascorbic acid required for half-maximal activation (K(a)). At a sinigrin concentration of 250 microM, the K(a) for ascorbic acid was 55 microM. Sulphate, a reaction product, was a competitive inhibitor of activity, having a K(i) of 60 mM with respect to sinigrin and of 27 mM with respect to ascorbate. Thus activation of myrosinase from R. sativus by ascorbic acid exemplifies an unusual and possibly unique example of linear 'uncompetitive activation' (i.e. a proportionate increase in V(max) and K(m)) of an enzyme. The enzyme also had beta-glucosidase activity and hydrolysed p-nitrophenyl-beta-d-glucopyranoside. (+info)
The binding motif recognized by HU on both nicked and cruciform DNA.
The heterodimeric HU protein, highly conserved in bacteria and involved in transposition, recombination, DNA repair, etc., shares similarity with histones and HMGs. HU, which binds DNA with low affinity and without sequence specificity, binds strongly and specifically to DNA junctions and DNA containing single-strand breaks. The fine structure of these specific complexes was studied by footprinting and HU chemically converted into nucleases. The positioning of HUalphabeta on nicked DNA is asymmetrical and specifically oriented: the beta-arm binds the area surrounding the break whereas the alpha-arm lies on the 3' DNA branch. This positioning necessitates a pronounced bend in the DNA at the discontinuous point, which was estimated by circular permutation assay to be 65 degrees. At junctions, HU is similarly asymmetrically positioned in an identical orientation: the junction point plays the role of the discontinuous point in the nicked DNA. The HU binding motif present in both structures is a pair of inclined DNA helices. (+info)
Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.
Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan. (+info)
Rescuing activity of galactoglycerolipids from cellular lesions induced by 5-aminolevulinic acid.
An anti-oxygen radical reagent of a bacterial metabolite, M874 monogalactoglycerolipid (di-O-12-methyl-tetradecanoyl-3-O-beta-D-galactopyranosyl-sn-glycerol ), was tested for its ability to protect two organisms against cellular lesions induced by 5-aminolevulinic acid (ALA) and light. In Corynebacterium flavescens ATCC 10340, extracellular uroporphyrin and coproporphyrin were the main porphyrin products. Although less than 2 mM ALA increased porphyrin synthesis, ALA levels above 3 mM inhibited the synthesis. Depending on the light intensity, the amount of porphyrin decreased and ALA-induced cytotoxicity increased. The lesion was more severe in the case of coproporphyrin than uroporphyrin. The porphyrin lesion produced in low intensity light (300 lx) was considerably reduced by 100 microM M874 glycolipid, although the reduction in intense light (3,000 lx) was restricted to a lower level. Similar results were obtained with radish (Raphanus sativus). The ALA concentration that inhibited porphyrin synthesis and stem growth was similar to that seen with C. flavescens. Although the exogenous addition of M874 glycolipid to the radish did not prevent ALA-induced cellular injury, the co-culture of radish and a glycolipid producing bacterium (Microbacterium sp. M874) resulted in a significant prevention of cellular injury. This was true only under enforced adhesion conditions through the action of a polysaccharide flocculant H12. Some species of monogalactoglycerolipids were found in Corynebacterium and radish that showed prominent oxygen radical-protecting activities similar to that of M874 glycolipid. These monogalactoglycerolipids might function in vivo as agents to prevent ALA-induced cytological lesions, although the concentrations were low in Corynebacterium and radish. (+info)
Rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen coevolution.
Many pathogen recognition genes, such as plant R-genes, undergo rapid adaptive evolution, providing evidence that these genes play a critical role in plant-pathogen coevolution. Surprisingly, whether rapid adaptive evolution also occurs in genes encoding other kinds of plant defense proteins is unknown. Unlike recognition proteins, plant chitinases attack pathogens directly, conferring disease resistance by degrading chitin, a component of fungal cell walls. Here, we show that nonsynonymous substitution rates in plant class I chitinase often exceed synonymous rates in the plant genus Arabis (Cruciferae) and in other dicots, indicating a succession of adaptively driven amino acid replacements. We identify individual residues that are likely subject to positive selection by using codon substitution models and determine the location of these residues on the three-dimensional structure of class I chitinase. In contrast to primate lysozymes and plant class III chitinases, structural and functional relatives of class I chitinase, the adaptive replacements of class I chitinase occur disproportionately in the active site cleft. This highly unusual pattern of replacements suggests that fungi directly defend against chitinolytic activity through enzymatic inhibition or other forms of chemical resistance and identifies target residues for manipulating chitinolytic activity. These data also provide empirical evidence that plant defense proteins not involved in pathogen recognition also evolve in a manner consistent with rapid coevolutionary interactions. (+info)
Modification of sorbitol MacConkey medium containing cefixime and tellurite for isolation of Escherichia coli O157:H7 from radish sprouts.
A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-beta-D-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate Escherichia coli O157:H7 from radish sprouts. Of 101 non-E. coli bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of E. coli O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were beta-galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of E. coli O157:H7 strains were colorless and beta-galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E. coli O157:H7. (+info)
Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance.
Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model. (+info)
Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis, and related genera (Brassicaceae).
We analyzed sequence variation for chalcone synthase (Chs) and alcohol dehydrogenase (Adh) loci in 28 species in the genera Arabidopsis and Arabis and related taxa from tribe Arabideae. Chs was single-copy in nearly all taxa examined, while Adh duplications were found in several species. Phylogenies constructed from both loci confirmed that the closest relatives of Arabidopsis thaliana include Arabidopsis lyrata, Arabidopsis petraea, and Arabidopsis halleri (formerly in the genus Cardaminopsis). Slightly more distant are the North American n = 7 Arabis (Boechera) species. The genus Arabis is polyphyletic-some unrelated species appear within this taxonomic classification, which has little phylogenetic meaning. Fossil pollen data were used to compute a synonymous substitution rate of 1.5 x 10 substitutions per site per year for both Chs and Adh. Arabidopsis thaliana diverged from its nearest relatives about 5 MYA, and from Brassica roughly 24 MYA. Independent molecular and fossil data from several sources all provide similar estimates of evolutionary timescale in the Brassicaceae. (+info)