RESUMEN
We studied the effects of new water-soluble polysubstituted fullerene C60 (PFD) derivatives on activity of Ca2+-Mg2+ ATPase of the sarcoplasmic reticulum and cGMP phosphodiesterase. All examined fullerene derivatives inhibited activity of both enzymes. For instance, PFD-I, PFD-II, PFD-III, PFD-V, PFD-IX, PFD-X, and PFD-XI in a concentration of 5×10-5 M completely inhibited hydrolytic and transport functions of Ca2+-ATPase. These compounds in a concentration of 5×10-6 suppressed active transport of calcium ions by 51±5, 77±8, 52±5, 52±5, 100±10, 80±8, and 100±10%, respectively, and inhibited ATP hydrolysis by 31±3, 78±8, 18±2, 29±3, 78±8, 63±7, and 73±9%, respectively, uncoupling the hydrolytic and transport functions of the enzyme. PFD-I noncompetitive and reversibly reduced activity of Ca2+-ATPase (Ki=2.3×10-6 M). All the studied fullerene derivatives (except for PFD-VII) inhibited cGMP phosphodiesterase by more than 80% in concentration of 10-4 M and higher and by more than 50% in concentration of 10-5 M. PFD-I is a non-competitive reversible inhibitor of cGMP phosphodiesterase (Ki=7×10-6 M). These results allow us to expect antimetastatic, antiaggregatory, antihypertensive and vasodilative activity of the studied compounds.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , Calcio/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Fulerenos/farmacología , Retículo Sarcoplasmático/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , ATPasa de Ca(2+) y Mg(2+)/aislamiento & purificación , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/aislamiento & purificación , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Fulerenos/química , Hidrólisis , Transporte Iónico/efectos de los fármacos , Cinética , Músculo Esquelético/química , Conejos , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/enzimologíaRESUMEN
We studied the effects of water-soluble cationic dinitrosyl iron complexes with thiocarbamide and its aliphatic derivatives, new synthetic analogs of natural NO donors, active centers of nitrosyl [1Fe-2S]proteins, on activities of Ca2+-ATPase of sarcoplasmic reticulum and cGMP phosphodiesterase. Nitrosyl iron complexes [Fe(C3N2H8S)Cl(NO)2]0[Fe(NO)2(C3N2H8S)2]+Cl- (I), [Fe(SC(N(CH3)2)2(NO)2]Cl (II), [Fe(SC(NH2)2)2(NO)2Cl×H2O (III), and [Fe(SC(NH2)2)2(NO)2]2SO4×H2O (IV) in a concentration of 10-4 M completely inhibited the transporting and hydrolytic functions of Ca2+-ATPase. In a concentration of 10-5 M, they inhibited active Ca2+ transport by 57±6, 75±8, 80±8, and 85±9% and ATP hydrolysis by 0, 40±4, 48±5, and 38±4%, respectively. Complex II reversibly and noncompetitively inhibited the hydrolytic function of Ca2+-ATPase (Ki=1.7×10-6 M). All the studied iron-sulphur complexes in a concentration of 10-4 M inhibited cGMP phosphodiesterase function. These data suggest that the studied complexes can exhibit antimetastatic, antiaggregation, vasodilatatory, and antihypertensive activities.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Compuestos Ferrosos/química , Nitrocompuestos/química , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/enzimología , Adenosina Trifosfato , Animales , Transporte Biológico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Compuestos Ferrosos/farmacología , Cinética , Nitrocompuestos/farmacología , Ratas WistarRESUMEN
RHAMM-selective peptides in a concentration of 10 µg/ml (2×10(-7) M) inhibited the growth of MDA-MB-231 breast cancer cells over 48 h. Treatment of cancer cells with RHAMM-selective peptides induced apoptosis and necrosis and increased caspase-3 activity (by 30%).
Asunto(s)
Antineoplásicos/farmacología , Proteínas de la Matriz Extracelular/genética , Fibroblastos/efectos de los fármacos , Receptores de Hialuranos/genética , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Necrosis/inducido químicamente , Oxazinas , Péptidos/síntesis química , XantenosRESUMEN
The neuroprotective action of hybrid structures based on fullerene C60 with attached proline amino acid has been studied. Hybrid structures contained natural antioxidant carnosine or addends with one or two nitrate groups. It has been shown that all studied compounds had antioxidant activity and decreased the concentration of malondialdehyde in homogenates of the rat brain. Compound 1, which contained the antioxidant carnosine, has been found to be the most effective antioxidant. All compounds except IV and V inhibited the activity of monoamine oxidase B, while compounds I-IV increased the activity of monoamine oxidase A. All investigated compounds inhibited glutamate-induced Ca2+ uptake into synaptosomes of the rat brain cortex. Compound III, containing two nitrate groups, has been found to be the most effective inhibitor. This compound caused a significant increase of the currents of AMPA receptors (AMPA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid).
Asunto(s)
Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Fulerenos/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Antioxidantes/química , Encéfalo/citología , Encéfalo/enzimología , Encéfalo/metabolismo , Calcio/metabolismo , Fulerenos/química , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Estructura Molecular , Monoaminooxidasa/metabolismo , Fármacos Neuroprotectores/química , Ratas , Receptores AMPA/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
The kinetics of interaction between organic nitrates (3,3-bis(nitroxymethyl)oxetane) and cysteine were evaluated by the rate of nitrite ion formation at various concentrations of reagents and pH. The activities of natural reducing agents, including cysteine, glutathione, and NADH, in generating the nitrite ion from organic nitrates (3,3-bis(nitroxymethyl)oxetane) were compared. Cysteine was shown to be the most potent reducing agent. Studying the effectiveness of nitrates (trinitroglycerol, 3,3-bis(nitroxymethyl)oxetane, and nicorandil) at a concentration of 3 mM showed that the rate of nitrite ion accumulation in the reaction with 10 mM cysteine is 1.66, 0.37, and 0.02 microM/min, respectively.
Asunto(s)
Cisteína/química , Éteres Cíclicos/química , Glutatión/química , NADP/química , Vasodilatadores/química , Oxidación-ReducciónRESUMEN
NO-donating ability of nitrosyl [Fe-S] complexes, namely, mononuclear dinitrosyl complexes of anionic type [Fe(S2O3)2(NO)2]-(I) and neutral [Fe2(SL1)2(NO)2] with L1=1H-1,2,4-triazole-3-yl (II); tetranitrosyl binuclear neutral complexes [Fe2(SL2)2(NO)4] with L2=5-amino-1,2,4-triazole-3-yl (III); 1-methyl-1H-tetrazole-5-yl (IV); imidazole-2-yl (V) and 1-methyl-imidazole-2-yl (VI) has been studied. In addition, Roussin's "red salt" Na2[Fe2S2(NO)4] x 8H2O (VII) and Na2[Fe(CN)5NO] x H2O (VIII) have been investigated. The method for research has been based on the formation of Hb-NO adduct upon the interaction of hemoglobin with NO generated by complexes I-VIII in aqueous solutions. Kinetics of NO formation was studied by registration of absorption spectra of the reaction systems containing Hb and the complex under study. For determination of HbNO concentration, the experimental absorption spectra were processed during the reaction using standard program MATHCAD to determine the contribution of individual Hb and HbNO spectra in each spectrum. The reaction rate constants were obtained by analyzing kinetic dependence of Hb interaction with NO donors under study. All kinetic dependences for complexes I-VI were shown to be described well in the frame of formalism of pseudo first-order reactions. The effective first-order rate constants for the studied reactions have been determined. As follows from the values of rate constants, the rate of interaction of sulfur-nitrosyl iron complexes (I-VI) with Hb is limited by the stage of NO release in the solution.
Asunto(s)
Hemoglobinas/química , Hierro/química , Óxido Nítrico/química , Compuestos Nitrosos/química , Azufre/química , Cinética , Modelos Moleculares , Soluciones , AguaRESUMEN
The back thermal cis-trans isomerization reaction of stilbazole betaine in the presence of metmyoglobin was studied. The catalytic effect of metmyoglobin heme on the back thermal cis-trans isomerization reaction of stilbazole betaine was observed.
Asunto(s)
Betaína/efectos de la radiación , Metamioglobina , Piridinas/efectos de la radiación , Estirenos/efectos de la radiación , Betaína/análogos & derivados , Betaína/química , Catálisis , Oscuridad , Piridinas/química , Estereoisomerismo , Estirenos/química , Temperatura , Rayos UltravioletaRESUMEN
The temperature dependence of the rate constant of photoinduced electron transfer in the modified eosin-myoglobin complex by monitoring of the phosphorescence quenching of eosin is measured. The values of electron transfer rate constants are equal 10(2) + 10(3) s-1 in the temperature region 150-200 K. The kinetics of relaxation of the maximum of the time-resolved phosphorescence spectra of eosin on apomyoglobin is measured in the same temperature range. The solvation relaxation of the time-resolved phosphorescence spectra is nonexponential. The characteristic times of the solvation relaxation are given 10(-2) + 10(-4) s-1, that correlate with the time of electron transfer in this system. It was observed the "acceleration" of the relaxation rate of the time-resolved phosphorescence spectra of eosin in metmyoglobin due to nonequilibrium photoinduced electron transfer. The role of the matrix dynamics in photoinduced electron transfer in proteins is discussed.
Asunto(s)
Eosina Amarillenta-(YS)/química , Metamioglobina/química , Animales , Transporte de Electrón , Hemo/química , Cinética , Luminiscencia , Fotoquímica , Temperatura , BallenasRESUMEN
Conception of the microsecond intramolecular protein dynamics with wide distribution of relaxation times is used for interpretation of published data on electron transfer kinetics in natural and chemically modified proteins. This is the evidence not only the unit static structure, but the dynamical organization of proteins as high organized molecular systems.
Asunto(s)
Metales/análisis , Proteínas/química , Termodinámica , Electrones , CinéticaRESUMEN
Quantitative estimation of basic factors determining the electron transfer rate constant between cytochrome c and inorganic metal complexes and electron exchange rate constant based on the theory of nonadiabatic electron transfer in polar media is presented.
Asunto(s)
Grupo Citocromo c/metabolismo , Transporte de Electrón , Cinética , Modelos Biológicos , Oxidación-ReducciónRESUMEN
Self-interaction of triplet-excited probes and interaction between the triplet-excited probes and a quencher (ferrocene) at 30 degrees C have been studied in Sarcoplasmic reticulum membranes (SR) and in dimiristoyl phosphatidylcholine liposomes by means of annihilated delayed fluorescence (ADF) registration. Perylene, 7,12-dimethyl benzanthracene and 4-(2-anthryl) butanoic acid have been used as triplet probes. An average time decay ADF of perylene and 4-(2-anthryl) butanoic acid in SR is equal to 4,2: 10(-5) s and 2,3 X 10(-5) s, respectively. Triplet states constant quenching of perylene by ferrocene is equal to 2,1 X 10(7) (mM/g of lipids)-1 X s-1 in SR and 4,5 X 10(7) (mM/g of lipids)-1 X s-1 in liposomes. This means that lipids in a liquid crystalline phase form continuous extended regions containing more than 7 X 10(3) lipid molecules and free of significant hindrances in probes diffusion.
Asunto(s)
Colorantes Fluorescentes , Retículo Sarcoplasmático , Fenómenos Químicos , Química Física , Membranas Intracelulares , Cinética , Lípidos de la Membrana , Proteínas de la Membrana , Modelos Biológicos , ViscosidadRESUMEN
The rate constants of exchange phosphorescence quenching of eosin by ferricyanide, hemin and hem-proteins (cytochrome c, myoglobin , hemoglobin, leghemoglobin) are measured. The steric factor of hem of cytochrome c is evaluated from the constant of eosin phosphorescence quenching by cytochrome c.
Asunto(s)
Hemo/metabolismo , Luminiscencia , Animales , Grupo Citocromo c/metabolismo , Hemoglobinas/metabolismo , Humanos , Cinética , Mioglobina/metabolismo , Oxidación-ReducciónRESUMEN
The temperature dependences of fluorescence and phosphorescence spectra maxima of chromophor labels--endogenic (tryptophan) and exogenic (eosinisothiocyanate)--were measured for the preparations of photosynthetic membranes and reaction centers from Rhodospirillum rubrum. It was found that the dipole mobility of protein-lipid matrix in the vicinity of the chromophores intensified markedly with a temperature rise from 150 to 300K resulting in the corresponding relaxation time tau r decrease from 10(0) to 10(-8) s. The efficiency of direct transfer of the photomobilized electron in the system of quinone acceptors (A1- leads to A2) of reaction centers (characteristic half-times of the process being 10(-3) divided by 10(-4) s) was shown also to increase sharply at temperatures higher than 200K parallel to the enhancement of molecular motions with tau r approximately 10(-8) s. Meanwhile, changes observed in the rate of recombination of primary photoproducts, i.e. an oxidized bacteriochlorophyll dimer, P+ and a reduced acceptor, A1- (characteristic half-time of 10(-1) divided by 10(-2) s) and the activization of low-frequency motions with tau r approximately 10(-3) s in the external layers and tau r less than 1 s in the internal parts of the reaction centers protein develop over the same range of low temperatures (150-220 K). The nature of interactions which determine the dependence of the photosynthetic electron transport on the molecular mobility of the membrane proteins is discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fotosíntesis , Rhodospirillum rubrum/metabolismo , Transporte de Electrón , Cinética , Complejos de Proteína Captadores de Luz , Mediciones Luminiscentes , Proteínas del Complejo del Centro de Reacción Fotosintética , Espectrometría de Fluorescencia/métodosRESUMEN
An analysis of spin,- fluorescence, triplet- and Mössbauer labeling techniques is given as well as the method of radical pair recombination for investigation of globular proteins molecular dynamics in the temperature range of 50-300 K and correlation time tau c = 10(-2) - 10(11) s-1. Based on experimental evidence and literature data a conclusion is drawn on the existence of protein microheterogeneity at negative temperatures. A dynamic model of a globular protein is proposed. It is supposed to be a system of tightly-packed polypeptide proteins blocks connected by hinge-like linkage with a few degrees of freedom. The system is sunk in viscous amorphous medium composed of superficial protein groups and bound water molecules. The correlation between the protein molecular dynamics and enzymes activities is discussed.
Asunto(s)
Conformación Proteica , Proteínas , Espectroscopía de Resonancia por Spin del Electrón/métodos , Matemática , Modelos Moleculares , Espectrometría de Fluorescencia/métodos , Análisis Espectral/métodos , TemperaturaRESUMEN
Registration of the annihilated delayed fluorescence kinetics of anthracene, 1,2-benzanthracene, 1,2,5,6-dibenzanthracene, 7,12-dimethylbenzanthracene, pyrene, 1,2-benzpyrene, 3,4-benzpyrene, perylene, and other aromatic compounds under laser excitation permitted investigation of the quenching of excited triplet molecules by hydrophobic quenchers (ferrocene, nitroxide radicals). A long life-time of the triplet excited states (10(-4)-10(-5) s) makes it possible to investigate the probe-quencher interactions in liposome and natural membranes at the probe concentration of 10(-6)-10(-7) M and the ratio of quencher/lipid = = 10(-4). The method enables to investigate localization of the enzyme active centre (hemes, non-heme iron, flavine) and lipid matrix organization in the lipid-protein complexes.
Asunto(s)
Colorantes Fluorescentes , Liposomas , Membranas/ultraestructura , Modelos Biológicos , Matemática , Relación Estructura-ActividadRESUMEN
It was shown that eosine and erythrosine are competing inhibitors of the Ca2+-Mg2+- dependent ATPase active center of sarcoplasmic reticulum. The eosine and erythrosine inhibition constants are equal to 1.4 x 10(-6) M and 1.1 x 10(-6) M, respectively. Nitroxide radicals of various hydrophobicity and K3Fe(CN)6 were used to compare the constants of triplet states exchange quenching of erythrosine in aqueous solution, in lecithine liposomes and in ATPase active center of sarcoplasmic reticulum. It was established that ATPase binding center was immersed into a liquid phase and was not connected with lipids. Mn2+ and Gd3+-ions, which are competing with Mg2+ and Ca2+ for binding sites in the enzyme active center, diminished the phosphorescence quenching time of eosine at 77K. This means that the ion binding sites are less than 12 A apart from ATP-binding center.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Retículo Sarcoplasmático/enzimología , Animales , Sitios de Unión , ATPasa de Ca(2+) y Mg(2+) , Óxidos N-Cíclicos/farmacología , Ferricianuros , Cinética , Músculos/enzimología , ConejosRESUMEN
Possibility of using phosphorescent probes for membrane investigations was analysed on lecithin liposomes and rat liver microsomes taken as an example. It was shown that one quencher molecule on 10(4) lecithin molecules is sufficient for experimental registration of diffusion-controlled quenching of erythrosine phosphorescence by stable nitroxide radicals. It is possible to study the diffusion processes with D = 10(-5) divided by 10(-9) cm2s-1. Application of quenchers of different polarity allows to make a conclusion that the phosphorescent probe erythrosine is localized in liposomes in the region of polar heads of phosphatidyl choline. It was determined from the rate of phosphorescence quenching by radicals that the membrane microviscosity in this region at 20 degrees C equals approximately 1 puas. The coefficient of erythrosine lateral diffusion in liposomes estimated from their self-quenching equals 1,1 x 10(-8) cm2s-1. In the microsome erythrosine is localized in hydrophobic parts of proteins and is not accessible for the quencher molecules.
Asunto(s)
Membranas Intracelulares/ultraestructura , Liposomas , Microsomas Hepáticos/ultraestructura , Fosfatidilcolinas , Animales , Cinética , Luminiscencia , Ratas , ViscosidadRESUMEN
The role of protein matrix in the process of charge photorespiration on the model of eosin-casein complex was studied. We have also studied the kinetics of the electron transfer reactions being photosensitized by free eosin and eosin sorbed on casein in the donor (cystein) - acceptor (methylviologen) system. When eosin is sorbed on protein a 10-fold increase of the probability relation of non-retrospective direct electron transfer to the restrospective one was observed.
Asunto(s)
Caseínas/metabolismo , Eosina Amarillenta-(YS) , Transporte de Electrón , Cinética , Luz , Consumo de Oxígeno , Unión ProteicaRESUMEN
The structure of eosin--casein complex was studied by triplet label method. Quantitative data on the quantum--mechanic exchange interaction between eosin centres and external quenchers were obtained. The dynamic state of water--protein matrix at -20 degrees to -180 degrees C with eosin as fluorescence and phosphorescence labels and natural chromophores of protein--tryptophane was studied.