Inhibition of dextransucrase by Zn2+, Ni2+, Co2+, and Tris(hydroxymethyl)aminomethane (Tris)

Initial rate kinetics of polysaccharide formation indicate that Zn2+, Ni2+, and Co2+ inhibit dextransucrase [sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5] by binding to two types of metal ion sites. One type consists of a single site and has a low apparent affinity for Ca2+. At the remaining site(s), Ca2+ has a much higher apparent affinity than Zn2+, Ni2+, or Co2+, and prevents inhibition by these metal ions. These findings are consistent with a two-site model previously proposed from studies with Ca2+ and EDTA. Initial rate kinetics also show that Tris is competitive with sucrose, but that, unlike Zn2+, Tris does not bind with significant affinity to a second site. This argues that there is a site which is both the sucrose binding site and a general cation site.     

Tris(hydroxymethyl)aminomethane inhibits calcium uptake in vascular smooth muscle.

This report demonstrates that the commonly used buffering agent Tris(hydroxymethyl)aminomethane (Tris) in concentrations of 5 and 30 mM inhibits calcium (Ca2+) uptake in both rat aortic and portal venous smooth muscle. The data indicates that total exchangeable Ca2+ in portal vein is reduced by about 35% in 5 or 30 mM Tris, while the intracellular exchangeable Ca2+ is not significantly altered. On the other hand, in aortic smooth muscle, while 30 mM Tris reduces total exchangeable Ca2+ by about 20%, intracellular Ca2+ is reduced by 44% in 5 mM Tris and by 55% in 30 mM Tris. The present studies, thus, reveal that Tris exerts significant inhibitory effects on exchangeability and transmembrane movement of Ca2+ in at least two different types of smooth muscle.     

Mechanism of irreversible inhibition of O2 evolution in photosystem II by Tris(hydroxymethyl)aminomethane.

The dark reaction of tris(hydroxymethyl)aminomethane (Tris) with the O2-evolving center of photosystem II (PSII) in the S1 state causes irreversible inhibition of O2 evolution. Similar inhibition is observed for several other amines: NH3, CH3NH2, (CH3)2NH, ethanolamine, and 2-amino-2-ethyl-1,3-propanediol. In PSII membranes, both depleted of the 17- and 23-kDa polypeptides and undepleted, the rate of reaction of Tris depends inversely upon the Cl- concentration. However, the rate of reaction of Tris is about 2-fold greater with PSII membranes depleted of the 17- and 23-kDa polypeptides than with undepleted PSII membranes. We have used low-temperature electron paramagnetic resonance (EPR) spectroscopy to study the effect of Tris on the oxidation state of the Mn complex in the O2-evolving center, to monitor the electron-donation reactions in Tris-treated samples, and to observe any loss of the Mn complex (forming Mn2+ ions) after Tris treatment. We find that Tris treatment causes loss of electron-donation ability from the Mn complex at the same rate as inhibition of O2 evolution and that Mn2+ ions are released. We conclude that Tris reduces the Mn complex to labile Mn2+ ions, without generating any kinetically stable, partially reduced intermediates, and that the reaction occurs at the Cl(-)-sensitive site previously characterized in studies of the reversible inhibition of O2 evolution by amines.     

Resurveying the Tris buffer solution: the specific interaction between tris(hydroxymethyl)aminomethane and lysozyme

An unusual phenomenon, the specific interaction between tris(hydroxymethyl)aminomethane (Tris) and lysozyme (LZM), was demonstrated for the first time by rapid screen analysis of interactions using a quartz crystal microbalance (QCM) biosensor. This phenomenon was also observed in a surface plasmon resonance (SPR) system. Further study using high-performance affinity chromatography (HPAC) confirmed this specific interaction between LZM and immobilized Tris with an apparent dissociation constant (K_D) of 6.7 × 10⁻⁵ M. Molecular docking was carried out to identify possible modes of binding between LZM and Tris linked to a binding arm. The estimated binding free energy was −6.34 kcal mol⁻¹, corresponding to a K_D of 2.3 × 10⁻⁵ M, which correlated well with the experimental value. Based on the docking model, the three hydroxyl groups of Tris form intermolecular H bonds with Asp52, Glu35, and Ala107 in LZM. This study reinforces the importance of buffer selection in quantitative biochemical investigations. For a lysozyme ligand binding study, it is better to avoid using Tris when the ligands under study are weak binders.     

Catalysis of hydrolysis and aminolysis of benzylpenicillin mediated by a ternary complex with zinc ion and Tris(hydroxymethyl)aminomethane

It was discovered that the combination of zinc ion and Tris in the pH range 7.5–10 is a very effective true catalyst for hydrolysis and aminolysis (by Tris) of benzylpenicillin. Both Cu²⁺ and Ni²⁺ ions were nonreactive in this system. The rate of loss of penicillin from solution was found to be a linear function of zinc ion at concentrations up to 10^?5M, while the dependence upon Tris concentration is maximal at about 0.02–0.03 M at each pH studied. At high penicillin concentrations saturation kinetics was observed. Product assays showed the major product to be a penicilloic acid at low Tris concentrations and N(penicilloyl)-Tris at high Tris concentrations. A mechanism is proposed which suggests that the reaction is mediated by a ternary complex in which the metal ion acts to bring the reactants (penicillin and Tris) into close proximity and to lower the pK_a of a Tris hydroxyl, creating a strong nucleophile. This mechanism also explains the results of the product assays and the lack of reactivity of the other metal ions tested. This reaction may be related to the mechanism for a known zinc-dependent β-lactamase.     

Effect of tris(hydroxymethyl)aminomethane on isolated sarcoplasmic reticulum vesicles.

The preincubation of isolated sarcoplasmic reticulum vesicles in Tris-Cl (pH 7.3) increases their (Ca²⁺ + Mg²⁺)-dependent adenosine triphosphatase activity and decreases their ATP-dependent Ca uptake capacity. These effects of Tris are dependent on the preincubation time and the Tris concentration; they are maximal below 10 μm Ca and decrease upon the increase of Ca concentration in the preincubation media, and they increase upon the increase of the preincubation pH. Differences in ATPase activity between preincubated and control vesicles are abolished by A23187 but not by carbonyl cyanide p-trifluoromethoxy phenyl hydrazone. The results suggest that: (i) Preincubation of the vesicles in Tris causes an increase of their permeability for Ca, or a membrane damage. (ii) Tris must diffuse within the vesicles to promote these effects. (iii) Ca prevents these effects by decreasing the membrane permeability for Tris. The basic findings were reproduced replacing Tris by imidazole.     

Density-gradient centrifugation in lithium metatungstate and tris-(hydroxymethyl)aminomethane phosphotungstate

New substances--lithium metatungstate (MTL) and tris-(hydroxymethyl)aminomethane phosphotungstate (PTT)--have been presented for density-gradient preparation. The buoyant densities of protein, RNA, DNA and some nucleoproteins were determined in solutions of these salts. Nucleic acids have been smaller buoyant density (1.1 g/cm3) than the proteins in contrast to CsCl-gradients. The protein in PTT solution have buoyant density 1.5 g/cm3 and in MTL solution 2.0-2,3 g/cm3. It was shown that MTL gradients allow to reach better resolution in nucleoprotein analysis than CsCl gradients.     

Degradation of Ferric Chelate of Ethylenediaminetetraacetic Acid by Bacterium Isolated from Deep-Sea Stalked Barnacle

Twenty strains of marine bacteria that degrade ferric chelate of ethylenediaminetetraacetic acid (Fe-EDTA) were isolated from among 117 strains collected from a marine environment. Among them strain 02-N-2, which was isolated from stalked barnacle collected from the deep sea in the Indian Ocean, had the highest Fe-EDTA degradation ability and was selected for further study. The strain showed high Fe-EDTA degradation ability at different seawater concentrations. In addition, the intact cells of this strain had the ability to degrade such metal-EDTAs as Ca, Cu, and Mg. The strain was an aerobic, gram-variable, rod-shaped organism. The results of various taxonomic studies revealed that the strain had significant similarity to Bacillus jeotgali JCM 10885^T, which was isolated from a Korean traditional fermented seafood, Jeotgal.     

Combined Action of Sulfamethoxazole, Trimethoprim, and Ethylenediaminetetraacetic Acid on Pseudomonas aeruginosa

Potentiation of the sulfamethoxazole-trimethoprim combination by ethylene-diaminetetraacetic acid inhibited, in vitro, four strains of Pseudomonas aeruginosa which were resistant to the sulfamethoxazole-trimethoprim combination alone.     

A note on the synthesis of 5'-AMP ester of tris (hydroxymethyl)aminomethane by rat liver plasma membrane.

The alcohol-AMP synthesizine enzyme of rat liver plasma membrane also synthesizes the 5'-AMP ester of tris(hydroxymethyl)aminomethane as judged by the use of [alpha-32P] ATP and [U-14C] ATP. This synthetic process may decrease significantly the concentration of ATP during incubation.     

Protection of DNA from high LET radiation by two OH radical scavengers,tris (hydroxymethyl) aminomethane and 2-mercaptoethanol

The effect of high linear energy transfer (LET) radiation on DNAin vitro, both in protective and non-protective environments was investigated. Two hydroxyl radical scavengers, tris(hydroxymethyl) aminomethane and 2-mercaptoethanol, were compared for their ability to protect SV40 DNA from radiation damage over a wide LET range. At comparable OH scavenging capacities, significant differences were found between these protective agents, indicating that other, radical scavenger-dependent processes affected the extent to which the DNA was protected. In general, a decrease in single-strand breaks (SSBs) relative to double-strand breaks (DSBs) was observed as LET increased. This effect was more pronounced when a radioprotector was present. Comparison of the relative biological efficiency (RBE) of radiation damage as LET increased showed a peak of DSB production in the mid-LET range. These data agree with measurements made by Christensen et al. (1972). An explanation for this increase in DSB production efficiency has been proposed based on the particle track structure of high-LET radiation.Correspondence to: G. Taucher-Scholz