Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea.

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of L-tryptophan by dimethylallyl diphosphate to form 4-(gamma,gamma-dimethylallyl)-L-tryptophan. The enzyme from mycelia of Claviceps purpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an alpha 2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for KM and Vmax were determined in the metal-free EDTA buffer (KMDMAPP, 14 microM; KML-tryptophan, 40 microM; Vmax, 215 nmol min-1 mg-1), 4 mM CaCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 17 microM; Vmax, 504 nmol min-1 mg-1), and 4 mM MgCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 12 microM; Vmax, 455 nmol min-1 mg-1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.     

Identification of fatty acids in submerged cultures of Claviceps species

Abstract Fatty acids were identified in the submerged mycelium of Claviceps purpurea, Claviceps paspali, Claviceps fusiformis and Claviceps sp. SD-58 by gas chromatography-mass spectrometry (GC-MS) and used for a chemotaxonomical study. A close relatedness was found between Claviceps sp. SD-58 and C. fusiformis . The composition of fatty acids in high-production mutants differed substantially from that of the parent strains. Fatty acid analysis is thus probably only applicable in the taxonomy of non-mutated isolates of Claviceps cultured under standard conditions.     

Some characteristics of the uptake of glutamine by corn scutellum

Slices of corn scutellum were used to study amino acid uptake, a natural function of this tissue. The uptake of glutamine was found to be inhibited by several monovalent cations. The accompanying anion did not affect the inhibition. Divalent cations stimulated glutamine uptake, particularly at high glutamine concentrations. The inhibition by monovalent cations was reversed by divalent cations.     

Some characteristics of Ca2+ uptake by yeast cells

Summary Experiments were performed to obtain information on: (i) the specific properties of Ca²⁺ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca²⁺ in yeast. These were obtained mainly by considering actual concentrations of Ca²⁺ in the medium after substracting the amounts bound to the cell. Ak _m of 1.9 m and aV _max of 1.2 nmol (100 mg·3 min)^–1 were calculated.The effects of some inhibitors and other cations on Ca²⁺ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca²⁺ uptake in yeast.The effects of Mg²⁺ on the uptake of Ca²⁺ agree with the existence of a single transport system for both divalent cations.The actions of Na⁺ and K⁺ on the transport of Ca²⁺ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.     

Increased tryptophan uptake into the brain in hyperammonemia

Hyperammonemia was provoked in rats by urease injection over three days. Tryptophan transport into the forebrain measured by the bolus injection technique was increased in hyperammonemic rats in comparison with pairfed controls. The concentration of the large neutral aminoacids, of tryptophan and of 5-hydroxyindole acetic acid were increased in the forebrain and brainstem. Probenecid administration led to a significantly higher accumulation of 5-hydroxyindole acetic acid in the forebrain of hyperammonemic rats. Since liver function was not impaired the data indicate that hyperammonemia in absence of hepatic insufficiency alters the carrier function for large neutral aminoacids at the blood brain barrier.     

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species.

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.     

Some autecological characteristics of early to late successional tree species in Venezuela

The breadth of the continuum concept of strategy with respect to succession was tested on 21 tree and shrub species common in either unlogged or logged stands, respectively, in the Forest Reserve of Caparo, Venezuela, by examining morphological, physiological and population characteristics. Based on a preliminary abundance analysis, `early', `mid' and `late' successional species as well as `generalists' were distinguished. Early successional species, i.e. Ochroma lagopus, Heliocarpus popayanensis and Cecropia peltata were similar in many autecological aspects, e.g. monolayered leaf arrangement, orthotropic architectural models, no adaptive reiteration, clumped distribution, but differed in gap association and distribution along a drainage gradient. Mid-successional species established themselves both in large and small gaps (> 300 m[sup2 ]; 80–300 m[sup2 ]) and showed a clumped to regular distribution pattern in logged areas; they exhibited more diverse crown and leaf characteristics than early successional species. Late successional species established themselves only in small gaps and understorey, and showed a regular spatial pattern in undisturbed areas. All late successional species displayed architectural models with plagiotropic lateral axes and showed a multilayered leaf arrangement. Adaptive reiteration was a common feature of late successional species which could be further subdivided into large, medium-sized and small trees, indicating different light requirements at maturity. Generalists were common treelet and shrub species in both disturbed and undisturbed sites where they are also capable of completing their life cycle. The light compensation point (LCP) of an individual plant was strongly influenced by its crown illuminance. Large late successional species showed the widest range of LCP values, reflecting the increasing light availability with increasing height in mature forest. On the basis of many autecological characteristics, it was found (i) that there is in fact a continuum of species strategies with respect to succession even among early and mid-successional species and (ii) that the latter group of species showed the widest breadth of autecological traits, reflecting the heterogeneous environment in which they establish and mature.     

Some characteristics of photosynthetic inorganic carbon uptake of a marine macrophytic red alga

Abstract Some characteristics of photosynthetic inorganic carbon uptake by Palmaria palmata, a marine red macroalga, have been measured under physiological conditions in artificial seawater. The apparent affinity of thallus for CO₂ [K_1/2(CO₂)] at pH 8.0 and 15°C was 21.4±3.0mmol m^?3 CO₂ under air, and 25.7±70mmol m^?3 CO₂ under N₂. The corresponding values of V_max were 2.98 ± 0.42 and 3.65±0.87 mmol O₂ evolved g Chr^?1 s^?l. The apparent K_m(CO₂) of isolated ribulose bisphosphate carboxylase was determined at pH 8.0 and 30 °C to be 30.2 mmol m^?3 CO₂, and the corresponding value of V_max was 19.67 μniol CO₂ g protein^?1 s^?1. The CO₂ compensation points of the thallus were measured in artificial seawater at pH 8.0 under air and N₂, using a gas-chromatographic method. The values were relatively low, rising from 10 cm³ m^?3 at 15°C, to 35 cm³ m^?3 at 25°C, but were not affected by the O₂ concentration. The lack of an effect of O₂ on photosynthesis and on compensation point indicates that there is little photorespiratory CO₂ loss in this macroalga. The high affinity of the thallus for CO₂, and the low CO₂ compensation concentrations, are consistent with the occurrence of bicarbonate uptake in this alga.