Biomedical subjects

Artur Krezel

Publications and source records attributed to Artur Krezel.

12 recordsLinked to original sources

PubMed2007

Different redox states of metallothionein/thionein in biological tissue.

Mammalian metallothioneins are redox-active metalloproteins. In the case of zinc metallothioneins, the redox activity resides in the cysteine sulfur ligands of zinc. Oxidation releases zinc, whereas reduction re-generates zinc-binding capacity. Attempts to demonstrate the presence of the apoprotein (thionein) and the oxidized protein (thionin) in tissues posed tremendous analytical challenges. One emerging strategy is differential chemical modification of cysteine residues in the protein. Chemical modification distinguishes three states of the cysteine ligands (reduced, oxidized and metal-bound) based on (i) quenched reactivity of the thiolates when bound to metal ions and restoration of thiol reactivity in the presence of metal-ion-chelating agents, and (ii) modification of free thiols with alkylating agents and subsequent reduction of disulfides to yield reactive thiols. Under normal physiological conditions, metallothionein exists in three states in rat liver and in cell lines. Ras-mediated oncogenic transformation of normal HOSE (human ovarian surface epithelial) cells induces oxidative stress and increases the amount of thionin and the availability of cellular zinc. These experiments support the notion that metallothionein is a dynamic protein in terms of its redox state and metal content and functions at a juncture of redox and zinc metabolism. Thus redox control of zinc availability from this protein establishes multiple methods of zinc-dependent cellular regulation, while the presence of both oxidized and reduced states of the apoprotein suggest that they serve as a redox couple, the generation of which is controlled by metal ion release from metallothionein.

Animals↗

PubMed2006

Sequence-specific Ni(II)-dependent peptide bond hydrolysis in a peptide containing threonine and histidine residues.

Previously we demonstrated that Ni(II) complexes of Ac-Thr-Glu-Ser-His-His-Lys-NH2 hexapeptide, representing residues 120-125 of human histone H2A, and some of its analogs undergo E-S peptide bond hydrolysis. In this work we demonstrate a similar coordination and reactivity pattern in Ni(II) complexes of Ac-Thr-Glu-Thr-His-His-Lys-NH2, its threonine analogue, studied using potentiometry, electronic absorption spectroscopy and HPLC. For the first time we present the detailed temperature and pH dependence of such Ni(II)-dependent hydrolysis reactions. The temperature dependence of the rate of hydrolysis yielded activation energy E(a) = 92.0 kJ mol(-1) and activation entropy DeltaS# = 208 J mol(-1) K(-1). The pH profile of the reaction rate coincided with the formation of the four-nitrogen square-planar Ni(II) complex of Ac-Thr-Glu-Thr-His-His-Lys-NH2. These results expand the range of protein sequences susceptible to Ni(II) dependent cleavage by those containing threonine residues and permit predictions of the course of this reaction at various temperatures and pH values.

Amino Acid Sequence↗

PubMed2006

Zinc-buffering capacity of a eukaryotic cell at physiological pZn.

In spite of the paramount importance of zinc in biology, dynamic aspects of cellular zinc metabolism remain poorly defined at the molecular level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concentration of 264 microM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not occupied by zinc, resulting in a surplus of 28 muM ligands (average Kd(c) = 83 pM) that ascertain cellular zinc-buffering capacity and maintain the "free" zinc concentration in proliferating cells at picomolar levels (784 pM, pZn = 9.1). This zinc-buffering capacity allows zinc to fluctuate only with relatively small amplitudes (DeltapZn = 0.3; below 1 nM) without significantly perturbing physiological pZn. Thus, the "free" zinc concentrations in resting and differentiated HT-29 cells are 614 pM and 1.25 nM, respectively. The calculation of these "free" zinc concentrations is based on measurements at different concentrations of the fluorogenic zinc-chelating agent and extrapolation to a zero concentration of the agent. It depends on the state of the cell, its buffering capacity, and the zinc dissociation constant of the chelating agent. Zinc induction of thionein (apometallothionein) ensures a surplus of unbound ligands, increases zinc-buffering capacity and the availability of zinc (DeltapZn = 0.8), but preserves the zinc-buffering capacity of the unoccupied high-affinity zinc-binding sites, perhaps for crucial physiological functions. Jointly, metallothionein and thionein function as the major zinc buffer under conditions of increased cellular zinc.

Cell Differentiation↗

PubMed2006

Synaptic release of zinc from brain slices: factors governing release, imaging, and accurate calculation of concentration.

Cerebrocortical neurons that store and release zinc synaptically are widely recognized as critical in maintenance of cortical excitability and in certain forms of brain injury and disease. Through the last 20 years, this synaptic release has been observed directly or indirectly and reported in more than a score of publications from over a dozen laboratories in eight countries. However, the concentration of zinc released synaptically has not been established with final certainty. In the present work we have considered six aspects of the methods for studying release that can affect the magnitude of zinc release, the imaging of the release, and the calculated concentration of released zinc. We present original data on four of the issues and review published data on two others. We show that common errors can cause up to a 3000-fold underestimation of the concentration of released zinc. The results should help bring consistency to the study of synaptic release of zinc.

Animals↗

PubMed2004

Acid-base versus structural properties of an aminoglycoside antibiotic--sisomicin: NMR and potentiometric approach.

Aminoglycoside antibiotics constitute a class of the drugs of high interest, whose therapeutic action is based upon the electrostatic interaction with the variety of RNA molecules. The positive charge of these drugs molecules, located at their amino functions, has a prevailing influence on this process. The potentiometry and (1)H NMR spectroscopy are applied hereby to achieve the characteristics of the acid-base properties of particular protonating groups. We found that the pK values of deprotonation processes cover a wide values range 6-9.8. The correlation spectra of sisomicin, both COSY and TOCSY, allowed attributing unambiguously individual signals to the corresponding protons. These spectra involve a lot of the cross-peaks originating from the B and C rings protons, while the analogous signals originating from A rings protons are less numerous. Molecular modeling provided that the methylated amino group of A ring is located too far from the protonated functions of the remaining rings to affect their pK values. The phenomena observed herein are discussed in line of strength of the analogous processes observed for other aminoglycosides. As the result, four types of amino groups consisted within these antibiotics are distinguished.

Acid-Base Equilibrium↗

PubMed2004

A formula for correlating pKa values determined in D2O and H2O.

A linear correlation between pH-meter readings in equivalent D2O and H2O solutions, determined experimentally, leads to a novel equation, which allows for a direct recalculation of pKa values measured in D2O into a H2O equivalent: pKH=0.929pKH*+0.42. The comparison of this equation with the previously used approach is discussed.

Deuterium↗

PubMed2004

Contrasting effects of metal ions on S-nitrosoglutathione, related to coordination equilibria: GSNO decomposition assisted by Ni(II) vs stability Increase in the presence of Zn(II) and Cd(II).

Complex formation between nitrosoglutathione (GSNO) and Zn(II), Cd(II), and Ni(II) ions was studied by potentiometry and spectroscopic techniques. GSNO forms simple ML and ML2 type complexes (L = GSNO) with these ions. The stability of GSNO in HEPES buffer solution, pH 7.4, increased in the presence of both Zn(II) and Cd(II), due to an indirect mechanism. A concentration-dependent destabilization of GSNO by Ni(II) ions was found to be linearly dependent on the NiL complex concentration. NiL forms ternary complexes readily. The NiLA- stoichiometry was found for l-His, and NiHLB3- and NiLB4- complexes were detected for GSSG as the second ligand. The formation of these complexes was found to inhibit GSNO decay, by limiting the concentration of the NiL complex. The mechanism of Ni(II)-assisted GSNO decomposition contains several steps, with a hypothetical ternary complex with GSH as a likely active form. These results provide experimental evidence for the stabilization of GSNO in solution by metal ions, which may provide an additional level of control and/or impairment of cellular redox signaling. The Ni(II)-dependent destabilization of GSNO may constitute a novel epigenetic mechanism in nickel carcinogenesis.

Cadmium↗

PubMed2003

Structure-function relationships in glutathione and its analogues.

The results are presented of measurements of protonation constants (potentiometry and NMR), UV spectroscopic properties and redox potentials of GSH and its five analogues, which are modified at the C-terminal glycine residue (gammaGlu-Cys-X, X = Gly, Gly-NH2, Gly-OEt, Ala, Glu, Ser). Strong linear correlations were found between various properties of the thiol and other functions of these peptides. These results allow discussion of the relationships between the structures and properties in glutathione and its analogues, and provide a novel chemical background for the issue of control of GSH reactivity.

Electrons↗

PubMed2003

Coordination properties of tris(2-carboxyethyl)phosphine, a newly introduced thiol reductant, and its oxide.

Acid-base properties and metal-binding abilities of tris(2-carboxyethyl)phosphine (TCEP), a newly introduced thiol group protectant, were studied in solution, using potentiometry, (1)H and (31)P NMR, and UV-vis spectroscopy, and also in the solid state by X-ray diffraction. Stability constants of complexes of the P-oxide of TCEP (TCEPO) were established by potentiometry. The list of metal ions studied included Ni(II), Cu(II), Zn(II), Cd(II), and Pb(II). Cu(II) catalyzed oxidation of TCEP to TCEPO. For all other systems ML complexes were found as major species at neutral pH with TCEP and TCEPO. Monoprotonated MHL species were also detected in weakly acidic conditions for all TCEP complexes and for the Pb(II) complex of TCEPO, while hydrolytic MH(-1)L complexes were found for TCEP at the weakly alkaline pH range. The NiL(4) complex was found to form at excess of TCEP. Overall, the complexes were found to be rather weak, with log beta(ML) values around 3-5 for TCEP and 1.5-2.5 for TCEPO. The phosphorus pK(a) value for TCEP, 7.68, suggests that it can be a good buffer for studies at physiological pH.

Journal Article↗

PubMed2003

May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex.

GSH and L-His are abundant biomolecules and likely biological ligands for Zn(II) under certain conditions. Potentiometric titrations provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favour of L-His (ca. 1 log unit of stability constant). The solution structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, determined by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calculations of competitivity of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(II) carrier.

Glutathione↗

PubMed2003

Correlations between complexation modes and redox activities of Ni(II)-GSH complexes.

The formation of Ni(II) complexes of GSH in conditions of 4-fold GSH excess over Ni(II) was studied by potentiometric titrations, UV-vis and CD spectroscopies, and magnetic susceptibility measurements. The following set of complexes was obtained in the pH range of 6-12: NiHL, Ni(2)L(2)(2)(-), NiHL(2)(3)(-), NiL(2)(4)(-), and NiH(-)(1)L(2)(5)(-). The first of these is an octahedral species, coordinated through the donors of the Glu moiety of GSH, while the remaining ones are largely square-planar, with participation of the thiol in Ni(II) coordination. Magnetic moments indicate the presence of a spin equilibrium for Ni(2)L(2)(2)(-), NiHL(2)(3)(-), and NiL(2)(4)(-) complexes. Phosphate ions apparently decompose the Ni(2)L(2)(2)(-) complex, converting it into a monomeric, high spin, ternary species. Among the molecular forms of GSH, HL(2)(-) is the one most susceptible to air oxidation, due to a presence of ionic interactions between its protonated amine and deprotonated thiol moieties. The complexation of Ni(II) accelerates air oxidation of GSH in alkaline solutions by a factor of 4, but this effect is absent at neutral pH. The damage to plasmid DNA by H(2)O(2) is facilitated by Ni(II) ions and inhibited by excess of GSH. However, the analysis of the concentration profile of this process indicates that octahedral Ni(II) complexes with GSH are involved in the formation of double strand breaks. Finally, numerical simulations of intracellular Ni(II) distribution, made possible by the determination of stability constants of Ni(II) complexes of GSH, indicate that histidine and ATP, rather than GSH, may act as ligands for Ni(II) in vivo. Altogether, our results suggest that the direct impact of GSH on Ni(II) toxicity may be of a limited character.

Air↗

PubMed2002

Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin.

A comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, respectively) and short peptide analogues was performed using potentiometry and spectroscopic techniques. It was found that while qualitative aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quantitative parameters (stability and rate constants) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modelling of Cu(II) and Ni(II) transport by albumins.

Animals↗