RESUMEN
Intrinsic tryptophan fluorescence and fluorescence of a hydrophobic probe bis-ANS were used to study the effect of phalloidin, a bicyclic heptapeptide toxin, on stability of monomeric (G) and polymeric (F) actin. It was found that bis-ANS fluorescence is sensitive to the actin polymerization process. Phalloidin in concentrations from 1 to 2 molecules per 1 actin molecule shifts the thermally induced unfolding transition in F-actin toward about 15 degrees C higher temperatures. The stabilizing effect of phalloidin is even more evident in the case of urea denaturation of F-actin. Moreover, phalloidin stabilizes against denaturing by not only F-actin, but G-actin as well, showing direct stabilizing interactions between phalloidin and G-actin.
Asunto(s)
Actinas/química , Faloidina/química , Naftalenosulfonatos de Anilina , Colorantes Fluorescentes , Desnaturalización Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.
Asunto(s)
Subfragmentos de Miosina/metabolismo , Nucleótidos/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Reactivos de Enlaces Cruzados , Polarización de Fluorescencia , Hidrólisis , Subfragmentos de Miosina/química , Pliegue de Proteína , Conejos , TemperaturaRESUMEN
Thermal denaturation of myosin subfragment 1 (S1) isoforms from rabbit skeletal muscle containing the different alkali light chains A1 and A2 [S1(A1) and S1(A2), respectively] were studied by various methods. Turbidity measurements showed that thermally induced (heating rate 1 degrees C min-1) aggregation of S1(A1) occurs at lower temperatures than that of S1(A2). However, the temperature dependences of the tryptophan fluorescence spectrum and that for ATPase inactivation were the same for S1(A1) and S1(A2). Thermal denaturation of the S1 isoforms was also studied by differential scanning microcalorimetry with the 'successive annealing' method. Three independently melting cooperative regions (domains) were revealed in the molecules of both isoforms. Heat sorption curves for the S1 isoforms were different only for the most thermolabile domain, which had a maximum at 36 degrees C for S1(A1) and at 40.5 degrees C for S1(A2). Two other peaks had maxima at 46-47 degrees C and 50-51 degrees C for both isoforms. It is proposed that alkali light chains A1 and A2 differently affect the conformation of the most thermolabile domain, which probably does not contain trytophan residues and does not take part directly in the formation of the active site of the S1 ATPase.
Asunto(s)
Isoenzimas/metabolismo , Músculos/metabolismo , Subfragmentos de Miosina/metabolismo , Miosinas/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Calor , Isoenzimas/química , Cinética , Peso Molecular , Subfragmentos de Miosina/química , Miosinas/química , Conejos , TermodinámicaRESUMEN
Thermal denaturation of myosin rod has been studied by differential scanning microcalorimetry and intrinsic tryptophane fluorescence methods. Use of the sequence annealing in the calorimetric measurement allows to decompose the total thermogram of rod into four elementary bands with maxima at 42, 46.5, 50 and 57 degrees C. Fluorescence changes occur at temperatures which coincide with the first, second and fourth calorimetric peaks. Changes of the time resolved and steady state fluorescence of myosin rod were interpreted using the data on localization of tryptophan residues in the molecule. The tryptophan fluorescence of myosin rod is assumed to monitor the denaturational changes in high meromyosin and probably in the hinge region but not in the subfragment 2.
Asunto(s)
Miosinas , Animales , Rastreo Diferencial de Calorimetría , Calor , Músculos/química , Conformación Proteica , Desnaturalización Proteica , Conejos , Espectrometría de Fluorescencia , TriptófanoRESUMEN
The structure of the myosin subfragment-1 (S1) from rabbit skeletal muscle was studied using differential scanning microcalorimetry. Three independently melting regions (domains) were revealed in S1. Selective denaturation of the middle 50 kDa segment of the S1 heavy chain resulted in the disappearance of the heat sorption peak corresponding to the melting of the first, the most thermolabile domain without any effect on the thermally induced blue shift of the intrinsic tryptophan fluorescence spectrum which occurs within the temperature region of melting of the second domain. It is concluded that the most thermolabile domain seems to correspond to the N-terminal part of the 50 kDa segment devoid of tryptophan residues.
Asunto(s)
Miosinas , Fragmentos de Péptidos , Animales , Calorimetría , Electroforesis en Gel de Poliacrilamida , Desnaturalización Proteica , Conejos , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The analysis of the intrinsic fluorescence parameters of T4 phage lysozyme in free state and in complex with inhibitor--disaccharide-tetrapeptide from the E. coli cell wall has been carried out. A comparison of the fluorescence changes with the results obtained by difference spectrophotometry and with the data of Elwell and Schellman on the intrinsic fluorescence of wild type WT and mutant eRI T4 phage lysozymes and a consideration of the three dimensional structure of the protein allows to represent the protein fluorescence parameters as a sum of contributions of the individual tryptophan residues. According to the proposed scheme Trp-126 does not emit neither in the free protein nor in the complex; the fluorescence parameters of Trp-158 (lambda m 332 nm, q = 0.27) are not affected by binding of the inhibitor, but all the fluorescence changes are due to the rise of the quantum yield (from 0.135 to 0.315) and the blue shift (from 332 to 328 nm) of the fluorescence of Trp-138.
Asunto(s)
Escherichia coli , Muramidasa/metabolismo , Peptidoglicano/metabolismo , Fagos T/enzimología , Triptófano/metabolismo , Muramidasa/antagonistas & inhibidores , Espectrometría de FluorescenciaRESUMEN
Kinetics of photoinduced reaction of HRP reduction by indolil acetic acid and dependence of the reaction rate and fluorescence of indoliol acetic acid on quenchers (I, Cst concentration was investigated. It was demonstrated that both I- and Cst action on the reaction differed from that on fluorescence. So the most probable reaction pathway is that through metastable triplet state of the substrate.
Asunto(s)
Peroxidasa de Rábano Silvestre , Ácidos Indolacéticos , Peroxidasas , Luz , NAD , Oxidación-ReducciónRESUMEN
When studying enzymic and fluorescence properties of myosin and DTNB-treated myosin in the presence of K+, Na+, Li+, NH4+, Ca2+ and Mg2+ cations the following results were obtained. By the intrinsic protein fluorescence techniques no essential structural changes of myosin molecule at the dissociation of the DTNB light chain and activation myosin ATPase in the presence of different cations were found. The decrease of K+-EDTA-, the increase of Mg2+-activated and the stability of Ca2+-activated myosin ATPase may be the result of the modification of SH1 or SH2 sulfhydryl groups when treating the DTNB myosin in our conditions. The different level of decrease of the K+- and NH4+-activated myosin. ATPase may be explained by the fact, that myosin sulfhydryl groups have different effects on the activation of its ATPase by these cations.
Asunto(s)
Ácido Ditionitrobenzoico , Miosinas , Nitrobenzoatos , Adenosina Trifosfato , Cloruro de Amonio , Animales , Calcio , Catálisis , Fenómenos Químicos , Química , Activación Enzimática , Litio , Magnesio , Potasio , Conejos , Espectrometría de FluorescenciaRESUMEN
It is shown that when myosin and heavy (HMM) and light (LMM) meromyosins are treated with concentrated solutions of neutral salts there is change in a number of parameters of the fluorescence spectra of these proteins. For myosin and HMM this change takes place in the region of the same concentrations of LiCl, MgDl2, KSCN where there occurs dissociation of the light chains of the myosin molecule. Change in the fluorescence characteristics of myosin and HMM in the presence of these salts may be caused by two effects: change in the native conformation of the myosin molecule and dissociation of its light chains. The effect of salts on LMM fluorescence is in good agreement with general theory of the influence of concentrated salts on macromolecules.