Biomedical subjects

H Kozlowski

Publications and source records attributed to H Kozlowski.

At least 19 recordsLinked to original sources

PubMed2004

Nickel(II) binding to Cap43 protein fragments.

Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein.

Amino Acid Motifs↗

PubMed2001

Structural characterization of Ru-bleomycin complexes by resonance Raman, circular dichroism, and NMR spectroscopy.

A series of spectroscopic techniques including absorption and CD spectra, resonance Raman spectra, and (1)H NMR as well as electrospray mass spectrometry have shown that Ru(II) ion binds to bleomycin, forming an equimolar complex, similarly to Fe(II), i.e., via the secondary amine nitrogen, the pyrimidine ring nitrogen, the deprotonated peptide bond nitrogen of the histydyl residue, and the histidine imidazole nitrogen, which are bound in the equatorial positions, and the alpha-amino nitrogen of beta-aminoalanine, which coordinates in the apical position above pH 7. The reaction of Ru(II)-BLM with O(2), H(2)O(2),or PhIO leads to formation of the oxy species in which only one oxygen atom is bound to metal ion. According to our data, the reaction of Ru(II)-BLM complex with oxygen species leads to different product than that suggested for Fe(II)-BLM. The formation of the BLM-Ru-O-Ru-BLM dimeric unit, similar to that found for sterically unhindered Ru porphyrins, seems to be the most likely.

Anti-Bacterial Agents↗

PubMed2001

Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity.

Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance.

Circular Dichroism↗

PubMed2001

Ni(II) and Cu(II) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR.

The tetradecapeptide containing the 10 aminoacid repeated sequence on the C-terminus of the Ni(II)-induced Cap43 protein, was analyzed for Ni(II) and Cu(II) binding. A combined pH-metric and spectroscopic UV-VIS, EPR, CD and NMR study of Ni(II) and Cu(II) binding to the blocked CH3CO-Thr-Arg-Ser-Arg-Ser-His-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-NH2 (Ac-TRSRSHTSEGTRSR-Am) peptide, modeling a part of the C-terminal sequence of the Cap43 protein, revealed the formation of octahedral complexes involving imidazole nitrogen of histidine, at pH 5.5 and pH 7 for Cu(II) and Ni(II), respectively; a major square planar 4N-Ni(II) complex (about 100% at pH 9, log K* = -28.16) involving imidazole nitrogen of histidine and three deprotonated amide nitrogens of the backbone of the peptide was revealed; a 3N-Cu(II) complex (maximum about 70% at pH 7, log K*=-13.91) and a series of 4N-Cu(II) complexes starting at pH 5.5 (maximum about 90% at pH 8.7, log K* = -21.39 for CuH(-3)L), were revealed. This work supports the existence of a metal binding site at the COOH-terminal part of the Cap43 peptide.

Amino Acid Sequence↗

PubMed2001

Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant.

D,L-Dithiothreitol (DTT), known also as Cleland reagent, is a thiol group protectant, used commonly in peptide and protein chemistry. Therefore, it is often added at high concentrations in preparations of proteins relevant to heavy metal biochemistry. The coordination of five of these metal ions, Zn(II), Cd(II), Pb(II), Ni(II) and Cu(I) to DTT was studied by means of potentiometric titrations, and UV-Vis and NMR spectroscopies. It was found that DTT forms specific and very stable polymeric and monomeric complexes with all of these metal ions, using both of its sulfur donors. The quantitative description of these complexes in solution and the solid state provides the basis for predictions of interference from DTT in studies of metal ion binding of thiol-containing biomolecules.

Chelating Agents↗

PubMed2001

Mitomycin antitumor compounds. Part 1. CD studies on their molecular structure.

The UV-Vis and circular dichroism (CD) spectra of several mitomycin antitumor compounds and some of their derivatives were analyzed in order to attribute the proper assignment to their electronic transitions. The lowest energy pi-->pi* transition was found to depend on the effect of the auxochromic group in the aromatic ring, whereas the three n-->pi* transitions, present at around 240, 400 and 560 nm, are related to the C(9)==O of the carbamoyl group and to the C(8)==O and the C(5)==O of the quinone, respectively. The chirality of the C(9) is responsible for the sign of the Cotton effect (CE) at around 240 nm, whereas the substituents of the chromophore for mitosane derivatives and the conformation of the carbamoyloxymethyl group at C(9) determine the CE sign of the (1)A-->(1)L(b) transition. When the aziridine ring was opened and mitosenes derivatives were obtained, CD spectra did not differ significantly among the compounds and the bands associated to the different transitions had similar Cotton effect. Our findings suggest that the differences in the CD spectra, observed between mitosanes and mitosenes, are probably related to the more rigid molecular structure of the mitosene derivatives and the different conformations in solution of the C(9) side chain.

Antibiotics, Antineoplastic↗

PubMed2000

Interaction of Ni(II) and Cu(II) with a metal binding sequence of histone H4: AKRHRK, a model of the H4 tail.

Chromatin proteins are believed to represent reactive sites for nickel binding. The unique structure of the N-terminal tail of histone H4 contains sites for post-translational modification close to a histidine residue capable of anchoring binding sites for metal ions. We have analyzed as a minimal model for the H4 tail, the blocked peptide CH(3)CO-AKRHRK-CONH(2) for nickel and copper binding. Ultraviolet-visible, circular dichroism, electron paramagnetic resonance and nuclear magnetic resonance spectroscopic analysis showed that histidine acts as an anchoring metal binding site. A 1N complex is formed between pH=5-7 and 4-6 for Ni(II) and Cu(II), respectively, while at a higher pH a series of 4N complexes are formed. Above pH 8, the 2N high-spin octahedral resulted in a 4N low-spin planar Ni(II) complex. The stability constants of the Cu(II) (3N, 4N) and Ni(II) (4N) complexes with the peptide model of the H4 were distinctly higher than those for a similar blocked peptide with a histidine in the fourth position. Significant shifts in the alphaproton region in the 1H NMR spectrum of the 4N Ni-complex showed that the conformation of the peptide had been dramatically affected following Ni(II) complexation.

Amino Acid Sequence↗

PubMed2000

Can the 1,5-disubstituted tetrazole ring modify the co-ordinating ability and biological activity of opiate-like peptides?

The copper(II) complexing ability and the biological activity of beta-casomorphin-7 tetrazole analogues have been investigated. Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been used to establish the thermodynamic stability, speciation and structure of Cu(II) complexes with YP-psi(CN4)-FPGPI-NH2 (1), YPF-psi(CN4)-AGPI-NH2 (2) and YPFP-psi(CN4)-GPI-NH2 (3). Comparison of the binding ability of the tetrazole analogues reveals that the most effective ligand for copper(II) is YPF-psi(CN4)-AGPI-NH2. The effectiveness of this ligand comes from its particular conformation suited for the Cu(II) 2N co-ordination mode in the physiological pH region. The ability of casomorphin tetrazole analogues to activate rat mast cells to histamine release in vitro in the presence of copper(II) has been studied.

Animals↗

PubMed1999

Effect of the tetrazole cis-amide bond surrogate on the complexing ability of some enkephalin analogues toward Cu(II) ions.

A study of the effect of the tetrazole moiety, a cis-amide bond surrogate, on the Cu(II) coordinating properties of oligopeptides is reported. The insertion of the tetrazole moiety psi (CN4) into the peptide sequence of [Leu5]enkephalin considerably changes the coordination ability of the ligand. Potentiometric and spectroscopic results indicate that if the tetrazole moiety is in a suitable position in the peptide chain, i.e. if it follows the third residue, an unusual stable CuH-1L species involving 4N coordination is formed in the physiological pH region. The tetrazole psi (CN4) ring provides one of these nitrogens. The data indicate that Cu(II) ions are strongly trapped inside a bent peptide backbone. However, the coordination mode involving the tetrazole ring nitrogen does not prevent the hydrolysis process under strongly basic conditions.

Amino Acid Sequence↗

PubMed1999

Solution structure of iron(III)-anthracycline complexes.

The interaction of Fe(3+) with the anthracycline anticancer drug idarubicin (Ida) was studied by absorption, CD, Mössbauer, and EPR spectroscopy. The formation of two major Fe(3+)-Ida complexes, labeled I and II, was observed. In complex I, Fe(3+) ion was bound to anthracycline at the {C(12)=O; C(11)-O(-)} coordination site. In complex II, two Fe(3+) ions were bound at sites {C(5)=O; C(6)-O(-)} and {C(12)=O; C(11)-O(-)}, respectively. Complex I was an equimolar monomeric species with a 1:1 Fe(3+):Ida stoichiometry (beta(1) = 4.8 x 10(11) M(-1)), whereas in complex II the anthracycline ligand was bridging two metal ions, alternatively bound to both anthracycline ring chelating sites with the assumption that the ratio of Fe(3+):Ida in complex II was 2:1 (beta(2) = 5.3 x 10(24) M(-2)). Alternatively, complex II may be oligomeric with Fe(3+):Ida = 1:1 and with each Fe(3+) bridging two Ida molecules. Our findings could be important in understanding the biological effects of the anthracycline-ferric complexes. Thus, providing information about the nature of the Fe(3+)-Ida system, we suggest that the formal 1:3 Fe(3+):anthracycline complexes, reported in the previous literature, could be a mixture of species I, II, and free ligand.

Antineoplastic Agents↗

PubMed1999

Correlation between the kinetics of anthracycline uptake and the resistance factor in cancer cells expressing the multidrug resistance protein or the P-glycoprotein.

Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins, the 170-kDa P-glycoprotein (Pgp) and the 190-kDa multidrug resistance-associated protein (MRP1). One possible pharmacological approach to overcome drug resistance is the use of specific inhibitors, which enhance the cytotoxicity of known antineoplastic agents. However, while many compounds have been proven to be very efficient in inhibiting Pgp activity only some of them are able to inhibit MRP1. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs with physicochemical properties favoring the uptake of the drug by the resistant cells. The intracellular drug retention influences its cytotoxic effect. The level of the intracellular drug content is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). In this work, the kinetics of drug uptake and the kinetics of active efflux of several anthracycline derivatives in both Pgp expressing K562/Adr cells and MRP1 expressing GLC4/Adr cells was determined. Our data have shown that in both cell lines there is no correlation between the resistance factor and the kinetics of drug efflux by these pumping systems. However, a very good correlation between the resistance factor and the kinetics of drug uptake has been established in both cell lines: the resistance factor decreases when the kinetics of drug uptake increases. This work has clearly shown that when the rate of transmembrane transport of anthracycline is high enough, the efflux mediated by the protein transporter is not able to pace with it. The protein transporter essentially operates in a futile cycle and the resistance factor is tending to one. It does not mean, however, that when the resistance factor is close to one the anthracycline is not transported by the pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

How Fe3+ binds anthracycline antitumour compounds. The myth and the reality of a chemical sphinx.

The interaction of Fe3+ with several anthracycline antitumour antibiotics has been reinvestigated. Absorption and circular dichroism (CD) measurements were carried out (i) in aqueous solution and (ii) in semi-aqueous MeOH to avoid the stacking of the anthracycline molecules. The Fe3+ binding to anthracycline was dependent on the metal-to-ligand molar ratio, antibiotic concentration, ionic strength, and pH. The formation of two major Fe3(+)-anthracycline complexes, I and II, was observed for all the drugs. These species differed in their coordination modes to the anthracycline ligands. Complex I was a monomeric species, where Fe3+ was bound to the anthracycline through the {C(11)-O-; C(12) = O} chelating site. In complex II, Fe3+ was also bound through the {C(5) = O; C(6)-O-} coordination site. Thus, the antibiotic ligand was acting as a bridge between two metal ions, forming oligomeric (or polymeric) structures. The different degree of association of the anthracyclines could be responsible for the reactivity of the metal ion. In fact, complexes I and II could constitute mononuclear, binuclear or polynuclear Fe3+ species depending on the competitive kinetics of both coordination and hydrolysis of the metal ion.

Anthraquinones↗

PubMed1996

1H-NMR and 13C-NMR investigation of complexes of Mn2+ with ocytocin analogues in (2H6)dimethylsulfoxide.

Several ocytocin analogues were synthesised by substitution of the Pro residue with sarcosine or N-methylalanine, the glutamine residue with threonine and one of the cysteines with 2-mercaptopropionic acid. All the derivatives were investigated by NMR in dimethylsulfoxide solutions and evidence was obtained for similar preferred conformations in the solution free state. All peptides were shown to form complexes with Mn2+ in solution by the strong paramagnetic effects experienced by several proton resonances. Two structures could be determined, one formed by peptides containing threonine and the other by the remaining peptides. The two structures were delineated by molecular modelling using the Mn(2+)-proton distances obtained by NMR as restraints.

Alanine↗

PubMed1996

Some aspect of the interactions of adriamycin with human serum albumin.

The interaction of adriamycin with human serum albumin (HSA) has been studied by absorption, CD, fluorescence spectroscopy, and quantitative precipitating HSA-antibody test. Our results demonstrate that adriamycin react with HSA and the binding to the protein molecule has a very distinct influence on the stability of ADR in aqueous solutions. The drug molecule binds protein as a monomer. The structural studies have shown the conformational change of HSA modified by adriamycin. The binding of ADR lowers the helicity of the native protein of ca. 15% and ca. 10% in the case of acHSA. The quantitative precipitating test supports distinct changes in the conformation upon ADR binding that decreases the ability of HSA to precipitate with its antibody.

Circular Dichroism↗

PubMed1995

Coordination ability of digalactosamine, and di- and trigalacturonic acids. Potentiometric and spectroscopic studies of Cu(II) complexes.

Potentiometric and spectroscopic (EPR, CD, and absorption spectra) data obtained for digalactosamine and di- and trigalacturonic acid with Cu(II) have shown that the di-sugar binding is usually less efficient than that of monomeric units while the tri-sugar can probably simultaneously use two terminal subunits to coordinate a metal ion. The latter result may have some relevance for metal binding by polysaccharides. All sugar ligands use amino or carboxylate functions as an anchor site, as in monomeric units. Bulky oligomeric ligands protect formation of the bis complexes. This causes the hydrolysis to be a dominant process at higher pH.

Carbohydrate Conformation↗

PubMed1995

Cu(II) binding by angiotensin II fragments: Asp-Arg-Val-Tyr-Ile-His and Arg-Val-Tyr-Ile-His. Competition between amino group and imidazole nitrogens in anchoring of metal ions.

Potentiometric and spectroscopic (absorption, circular dichroism and electron paramagnetic resonance) study on the coordination of two angiotensin II fragments (Asp-Arg-Val-Tyr-Ile-His and Arg-Val-Tyr-Ile-His) to Cu(II) ions has shown that competition between amino and imidazole nitrogens to anchor metal ions is a complicated process and may lead to formation of macrochelate rings. The important factor that influences this competition is the distance between competing His and N-terminal residues (number of spacer residues in a peptide sequence).

Amino Acid Sequence↗

PubMed1995

Specific binding of Cu(II) ions by leucine-enkephalin analogs.

Results are reported from a potentiometric and spectroscopic (UV-visible, CD, and ESR) of the protonation constants and Cu(II)-complex stability constants of leucine enkephalin amide (H-Tyr-Gly-Gly-Phe-Leu-NH2, Leu-EN-amide) and two nitro analogs having 4-nitro substituent on the phenyl ring of the Phe residue, (H-Tyr-Gly-Gly-Phe(NO2)-LeuNH2, Leu-EN(nitro)- amide) and the other one with a sarcosine residue replacing the Gly3 residue (Leu-ENSar-amide). Over the pH range of 6-8.5, Leu-EN-amide interacts more strongly with Cu(II) than does the methionine analog, forming a more stable complex with three nitrogens coordinated. The Sar residue acts as a "breakpoint" to the formation of 3N or 4N complexes and, as a result, causes the formation of dimeric complexes bonded through the amino-N, a deprotonated peptide-N- and deprotonated Tyr-O- donors.

Amino Acid Sequence↗

PubMed1994

A dramatic change in the interaction of Cu(II) with bio-peptides promoted by SDS--a model for complex formation on a membrane surface.

The extent of complex formation between Cu(II) and many biologically active oligopeptides has been shown to change significantly in the presence of SDS micelles, a recognized model for cell lipid membranes. Protonation constants of peptides can be increased by up to 2 log unit, especially when they contain hydrophobic side chains. Metal complex formation is generally less extensive and the conformations of peptides can be altered dramatically when compared to those in simple aqueous solution.

Amino Acid Sequence↗