Relation of cell wall peroxidase activity with growth in epicotyls of Cicer arietinum. Effects of calmodulin inhibitors

Sánchez, O.J., Pan, A., Nicolás, G. and Labrador, E. 1989. Relation of cell wall peroxidase activity with growth in epicotyls of Cicer arietinum. Effects of calmodulin inhibitors.
Peroxidases are bound ionically to cell walls in epicotyls of Cicer arietinum L. cv. Castellana. The cell wall peroxidase activity increases during the growth of epicotyls, being the lowest in 3-day-old epicotyls with high growth capacity. The cell wall phenolic compounds, postulated natural substrates of cell wall peroxidases, also increase during growth.
The calmodulin inhibitors chlorpromazine and trifluoperazine decrease the elongation rate of epicotyls of Cicer arietinum. These inhibitors also cause an increase in the cell wall peroxidase activity and in the level of phenolic compounds. A possible regulatory effect of calmodulin on peroxidase activity is postulated.     

Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6₅22 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P2₁2₁2₁, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.     

Key role of the medium in methemalbumin-catalyzed peroxidation of aromatic free radical scavengers and antioxidants

Participation of the complexes of hemin and albumins (or delipidated albumins) in peroxidation of aromatic free radical scavengers and antioxidants was studied at varying hemin/albumin ratios. The radicalscavenging amines includedo-phenylenediamine (OPD) and tetramethylbenzidine (TMB); the antioxidants were gallic acid (GA) and GA polydisulfide (GAPD). Peroxidation reactions were carried out in buffered physiological saline (BPS) supplemented with 2% dimethylsulfoxide (DMSO), pH 7.4 (medium A), or in 40% aqueous dimethylformamide (DMF), pH 7.4 (medium B). In all systems involving methemalbumins, kinetic constants (k_cat), Michaelis constants(K _M), and the ratios thereof(k _cat/K_M) were determined for OPD oxidation in medium A and TMB oxidation in medium B. Oxidation of OPD, GA, and GAPD in medium A was characterized by a decrease in the catalytic activity of hemin after the formation of hemin-albumin complexes. Conversely, oxidation of TMB and OPD in medium B was distinguished by pronounced activation of hemin present within methemalbumins.     

The yeast HAL1 gene improves salt tolerance of transgenic tomato

Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K(+) concentration and decreasing intracellular Na(+) during salt stress. In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation. A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo. Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene. In addition, measurement of the intracellular K(+) to Na(+) ratios showed that transgenic lines were able to retain more K(+) than the control under salt stress. Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast.     

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5 mM dopachrome the oxygen consumption rate of TrT on 8 mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.     

Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers

On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQwl, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DRI-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1 . In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQwl-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.     

Linkage disequilibrium between cystic fibrosis and linked DNA polymorphisms in Italian families: A collaborative study

The locus D7S23 includes a CpG-enriched methylation-free island that maps midway between the markers J3.11 and met and is genetically very close to the mutation causing cystic fibrosis (CF). We have studied the linkage disequilibrium between four polymorphic markers from this locus (KM.19, CS.7, XV-2c, and PT-3) and the CF mutation (CF) in 127 Italian families. Strong linkage disequilibrium is found between KM.19, CS.7, and CF, and weaker but significant disequilibrium is found between XV-2c, PT-3, and CF. The disequilibrium between markers and CF for the Italian population provides additional information on the origin and homogeneity of the CF defect. This panel of probes is sufficiently informative to permit accurate prenatal diagnosis of CF in most families with an affected person, and the disequilibrium also allows indirect carrier detection/exclusion in some cases.