Search

The enzyme aspartate aminotransferase has been isolated from the halophilic bacterium Haloferax mediterranei in its apoenzyme form. The interaction with its coenzyme (pyridoxal phosphate) has been investigated. For concentrations up to 0.05 mᴍ, the incubation with pyridoxal phosphate reconstituted the active complex (holoenzyme) following a second order kinetic with a k2 of 5.2 min-1mᴍ-1. This active complex showed a dissociation constant (Kd) of 7.8 x 10-6 ᴍ. For concentrations higher than 0.1 mᴍ, pyridoxal phosphate produced an inactivation process with a complex second order kinetic. This inactivation is partially reverted by dialysis or by lysine treatment. Thus, after 80% of inactivation, 55% of the original activity is recovered by a long-time dialysis, and with 50 mᴍ lysine also a partial reactivation (among 20-33%) is observed. The enzyme treated with 1 mᴍ pyridoxal phosphate has a different behavior in Sepharose chromatography indicating that the modified enzyme presents a smaller size due to a conformational change.

Aspartate aminotransferase (AspAT, EC 2.6.1.1) from the halophilic archaebacterium Haloferax mediterranei was purified [Muriana, Alvarez-Ossorio and Relimpio (1991) Biochem. J. 278, 149-154] and further characterization of the effects of temperature on the activity and stability of the halophilic AspAT were carried out. The halophilic transaminase is most active at 65 degrees C and stable at high temperatures, under physiological or nearly physiological conditions (3.5 M KCl, pH 7.8). Thermal inactivation (60-85 degrees C) of the halophilic AspAT followed first-order kinetics, 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures. The salt concentration affected the thermal stability of the halophilic transaminase at 60 degrees C, suggesting that disruption of hydrophobic interactions may play an important role in the decreased thermal stability of the enzyme.

Nitrate reductase is induced in cells of Haloferax mediterranei by the presence of nitrate upon anaerobic conditions. This enzyme was purified more than 35-fold with a yield of 49%. Densitograms of polyacrylamide gel electrophoresis show the preparation to be 85% purity. The best enzyme preparation has a specific activity of 13.6 U /m g protein. It is the first halophilic nitrate reductase that has been purified near to homogeneity. The purification consists of five steps: an ammonium sulphate precipitation and four successive gel chromatographies with Sepharose CL-4 B, calcium phosphate, DEAE-Sephacel and Sephacryl S-200. An average Mr of 170,000 was estimated by gel chromatography and non-denaturing gel electrophoresis. Effectiveness of electron donors, cofactors and inhibitors are reported. At low salt concentration the halophilic nitrate reductase was inactivated following first-order kinetics. The Km for nitrate depends on salt concentration and shows values in the range from 2.5 to 6.7 mM.

The aspartate aminotransferase from Haloferax mediterranei, which is in the cell mainly as apoenzyme, requires high concentrations of salt for both activity and stability. The maximum activity is reached with 3.5 ᴍ KCl in the assay. The effect of different cations and anions has been studied using several types of salts. Monovalent cations show a significant difference in effectiveness of promoting the activity with the following order: K+ > Rb+ > Na+ > NH4 +. Mg++ and polyvalent cations with organic character cause partial activity with maximum effectiveness at 0.1 ᴍ, an inhibition at higher concentration is observed. Anions, added as potassium salts, promote enzyme activity with the following order: Cl- > NO3 - > I- > SCN-. Like activity, the enzyme stability depends on salt concentrations. Incubation of the enzyme with a low salt concentration leads to inactivation following pseudofirst order kinetics. The inactivated enzyme is partially reactivated by high concentrations of KC1 following second order kinetics. Taking into account the dimeric structure of this enzyme, high concentrations of salt could stabilize the dimer, which is the active form. The salt effects on halophile aspartate am inotransferase are discussed considering hydrophobic and electrostatic interactions.

An experimental system has been devised for the optimum aerobic growth and induction of the halophilic aspartate aminotransferase of an archaebacterium Haloferax mediterranei incubated in the presence of L-aspartate. H. mediterranei utilizes both L-aspartate and yeast extract as a sole or combined energy source. The maximum synthesis of the enzyme was promoted by 1% L-aspartate. The relationship between holo and apo forms of the halophilic transaminase, as the mechanism of the induction system, seems to be related to unusual regulatory properties.

Partially purified glutamine synthetase from rat liver underwent rapid inactivation upon incubation with NADH and benzyl viologen, under aerobic conditions. This in vitro inactivation was prevented by catalase or chelating-agents, which suggests that hydrogen peroxide and metal ions are involved in the process. Similar inactivation was observed when the rat liver glutamine synthetase was preincubated, under anaerobic conditions, with NADH and benzyl viologen, and hydrogen peroxide was added to the reaction mixture. A radical scavenger, histidine, partially prevents the inactivation, while hydrogen peroxide shows a low inactivation capacity when incubated without NADH. Furthermore, the fact that the inactivation can also be catalyzed by a model consisting of ferrous ions and hydrogen peroxide leads to the conclusion that hydroxyl radicals, or something with similar reactivity, are most likely produced through a Fenton reaction.

Aspartate aminotransferase from the archaebacterium Haloferax mediterranei was purified and found to be homogeneous. An average Mr of 66,000 was estimated. The native halophilic transaminase exhibited no maximum absorption at 410 nm, which indicates that the apo form is obtained by our purification procedure, and the molar absorption coefficient at 275 nm in 3.5 M-KCl (pH 7.8) was found to be 78.34 mM-1.cm-1. Plots of titration data show that 1 mol of halophilic aspartate aminotransferase binds 2 mol of pyridoxal 5′-phosphate. The halophilic transaminase behaved as a dimer with two similar subunits and had a maximum activity in the pH range 7.6-7.9 and at 65 degrees C in 3.5 M-KCl. By differential scanning calorimetry, the denaturation temperature of the halophilic holo- and apo-transaminase was determined to be 78.5 and 68.0 degrees C respectively at 3.3 M-KCl (pH 7.8). At low salt concentration the halophilic transaminase was inactivated, following first-order kinetics. The Km values for 2-oxoglutarate and L-aspartate, in 3 M-KCl (pH 7.8), were 0.75 mM and 12.6 mM respectively.

1. A series of ethyl- mono- and disubstituted phenyl-methylphosphonates have been synthesized and their biological activities in vitro and in vivo studied. 2. The treatment ϱ-σ-π of biological activity in vivo leads to the conclusion that the electronic effects of the para substituents play a preponderant role on the activity, whereas the role of the meta substituents is moreover conditioned by the steric factors. 3. The study of the effects that the solubility exerts on the activity has been shown to be too small to represent an appreciable factor. 4. The results obtained have been compared with their diethyl-phosphate analogues.

Administration orale de 10, 20, 40 et 60 mg/100 g de poids d'atrazine a des rats pendant 14 jours et examen des taux de sodium, potassium et chlorures dans l'urine, et aussi des taux de creatinine et d'albumine. Examen de l'activite de la lactate deshydrogenase et de l'α-hydroxybutyrate deshydrogenase dans le serum. Analyse des effets nephrotoxiques de l'atrazine

Phosphypyridoxyl trifluoroethylamine has been synthesized as an active site-directed 19F NMR probe for aspartate transaminase. This coenzyme derivative adds stoichiometrically to the apotransaminase as observed by both fluorescence and circular dichroism measurements. The fluorinated phosphypyridoxamine derivative, when bound to the apotransaminase, will not dissociate upon extensive dialysis or passage through Sephadex G-25. The compound behaves as a pyridoxamine phosphate derivative and not as a coenzyme-substrate complex, since both competing anions and dicarboxylic acid inhibitors still bind to the phosphopyridoxyl trifluoroethylamine enzyme. The 19F NMR spectra of the enzyme-bound phosphopyridoxyl trifluoroethylamine were measured as a function of pH, ionic strength, and temperature. The 19F MNR of the enzyme-bound coenzyme derivative revealed no predetermined asymmetry in the subunits of aspartate transaminase insolution in terms of differences in chemical shift or resonance line shape between the two environments. A pH-dependent chemical shift change of the single 19F resonance was observed, which is consistent with the influence of a single ionization with an apparent pKa of 8.4 in 0.10 M KCl at 30 degrees. Increasing the ionic strength resulted in increasing values for the observed pKa, the highest recorded value was 9.1 in 3.0 M KCl. The temperature dependence of the pH titration of the chemical shift gives deltaH' of ionization of 10.5 kcal/mol. The evidence suggests a possible epsilon-amino group, electrostatically affected by positive charges, being responsible for the titration effect of the active site-bound fluorine derivative of pyridoxamine phosphate.

Trifluoromethionine and p and o -monofluorophenylalanine can be accepted by cytoplasmic aspartate transaminase as substrates; α-trifluoromethyl-DL-alanine can act as an inhibitor of the enzyme. Covalent pyridoxal phosphate (PLP) derivatives of these amino acids can be prepared and after reduction of the resulting Schiff's base with NaBH4 the isolated PLP-fluorinated amino acids used as inhibitors of apoenzyme. The union of the fluorinated-PLP compounds to apoenzyme is stoichiometric as judged by fluorescence and circular dichroism techniques as well as by inhibition of enzymatic transamination with the natural substrates, aspartate and α-ketoglutarate. The transaminase retains its stereospecific discrimination at the substrate level and when presented with a racemic mixture of fluorinated (DL) amino acid-PLP compound accepts only one isomer. The holoenzyme-fluorinated substrate complexes are stable to dialysis and chromatography through Sephadex G-25, yet, they can be resolved by phosphate buffers into native apoenzyme and fluorinated amino acid-PLP compound.

Several groups of rats were treated with dipyridamol and dihydroergotoxine methane sulphonate and their association. Their clinical constants were then monitored and their brain tissue examined by optical microscopy. This study indicates that the association has a remarkable effect on the pO2, maintaining the level of 2,3-diphosphoglycerate concentration, and moreover a vasodilatory action, facilitating a better supply of oxygen to the cerebral cells. The results show the advantage of these drugs in treating cerebral vascular disease.

This paper describes an examination of the cell envelope stability opposite to disruption by chemical and physical methods of extremely halophilic bacteria. The following methods of cell treatment were studied: solvent and chelating agents; pressure shearing at several pressures; ultrasonic disintegration for various times; ballistic disintegration; grinding with cold alumina; lysozyme digestion; osmotic shock; and freezing and thawing. The procedure is based on the determination of three cytoplasmic enzymes released by the cell treatment. Menadione reductase was also used as convenient marker enzyme for damage to the permeability barrier. Of all the methods, only pressure shearing and ultrasonic disintegration yielded a crude extract with high halophilic enzyme activities. These procedures are suitable in designing a cell fractionation scheme for halophilic enzyme purifications.

Thirty derivates from substituted-phenyl-ethyl methylphosphonates have been synthesized and their inhibiting power of acetyl-cholinesterase have been examined in vitro and in vivo. The correlation between inhibition of the enzyme and electrophylic power of the substituent of the phenyl group was excellent, but when this group contains two substituents, steric factors appear to operate. The activity of these compounds has been demonstrated to be higher than their phosphate analogs.

The effects of monovalent and divalent cations on the cell viability of an extremely halophilic bacteria H. mediterranei were examined. High magnesium concentrations (≧72 mM) were required for stabilization of H. mediterranei cells, although calcium ions were more potent than magnesium in preserving viability. On the other hand, H. mediterranei cells are also able to survive in the presence of monovalent cations, such as ammonium and potassium, at least 14 days. The implications for maintaining stability and neutrality of several cell envelope components by monovalent and divalent cations are discussed.

The binding of succinate and perfluorosuccinate to the supernatant and mitochondrial isoenzymes of aspartate transaminase has been studied using 1H and 19F nuclear magnetic resonance (NMR) as probes. The binding was measured for the pyridoxal (El) and pyridoxamine (Em) forms of the holoenzyme, the apoenzyme, and the pyridoxal form after reduction of the internal Schiff's base with NaBH4 (reduced holoenzyme). The NMR parameters studied were band broadening or chemical shift changes upon dicarboxylic acid binding to the enzyme. The dissociation constant values of succinate and perfluorosuccinate for the active forms of each isozyme, El and Em, are in the 1 to 4 mm range. Kd values of the inactive forms of the supernatant transaminase for perfluorosuccinate are 18.3 mm, reduced form, and 12.0 mm, apoenzyme form. Anions are competitive with dicarboxylic acid binding and, because of mutual exclusion, do not alter the correlation time, τc of bound succinate. This competition allows for the calculation of the chloride affinity of the pyridoxal, Kd = 9 mm (supernatant), 3 mm (mitochondrial), and pyridoxamine forms, Kd = 5 mm (supernatant), 1.6 mm (mitochondrial), of each isoenzyme. Because amino acids and glutarate also compete with perfluorosuccinate binding in the El, Em, and apoenzyme forms, it is concluded that dicarboxylic acids bind to these forms of the enzyme and that amino acids can form complexes with the pyridoxamine form. The correlation time of bound dicarboxylic acid shows an increase, i.e. more restricted segmental motion of succinate, with decreasing pH and is particularly critical in the pH 5 and pH 8 regions. The fastest correlation time of perfluorosuccinate (greatest segmental motion) corresponds to that bound to the reduced holoenzyme. The above effects agree with the competition-pH dependence between dicarboxylic acids and anions. Since there is evidence of the active center histidyl residue as the anion binding site (Cheng, S., and Martinez-Carrion, M. (1972) J. Biol. Chem. 247, 6597–6602) and of the Schiff's base nitrogen as the low pH ligand of a carboxyl group in glutaric acid (Jenkins, W. T., and D'Ari, L. (1966) J. Biol. Chem. 241, 5667), a proposal for the binding of dicarboxylic acids is made. Dicarboxylic acids inhibit because they form a bridge between the active center histidyl residue and the pyridoxal phosphate internal aldimine with the e-amino group of a lysyl residue. Thus, the binding subsites for a competitive inhibitor such as succinate, although structurally similar to the substrates, differ from the productive mode of binding of the amino or keto acids which form covalent enzyme-substrate complexes.