RESUMO
Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed "reactive" astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.
Assuntos
Astrócitos/efeitos dos fármacos , Metalotioneína/farmacologia , Regeneração/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/fisiologia , Axônios/efeitos dos fármacos , Axônios/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Metalotioneína/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/fisiologia , Ratos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.
Assuntos
Cádmio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Zinco/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Cádmio/química , Humanos , Concentração de Íons de Hidrogênio , Metalotioneína/química , Metalotioneína/isolamento & purificação , Metalotioneína/metabolismo , Metalotioneína 3 , Proteínas do Tecido Nervoso/química , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Zinco/químicaRESUMO
The reaction of Cd5Zn2-metallothionein (MT) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temperature conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate constants obtained were independent from the concentration of Nbs2 but were linearly dependent on the concentration of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the observed rate constant increases. The reactivity of MT towards Nbs2 has been probed at 4 microM concentration of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8 +/- 2.7 kJ.mol-1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a number of nonmetallic ligands moderately accelerate the reaction of MT with Nbs2 and hyperbolic dose-response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding constants were calculated as follows: ATP, K = 0.31 +/- 0.06 mM; ADP, K = 0.26 +/- 0.07 mM. Several compounds such as AMP, S-methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concentrations.
Assuntos
Ácido Ditionitrobenzoico/metabolismo , Metalotioneína/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cádmio/metabolismo , Quelantes/farmacologia , Ácido Edético/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Fígado , Fosfatos/metabolismo , Ligação Proteica , Coelhos , Temperatura , Termodinâmica , Zinco/metabolismo , Zinco/farmacologiaRESUMO
Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.
Assuntos
Cobre/química , Metalotioneína/química , Cádmio/química , Dicroísmo Circular , Cinética , Espectroscopia de Ressonância Magnética , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.
Assuntos
Intestinos/química , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Animais , Calbindinas , Cálcio/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Desnaturação Proteica , Resinas Sintéticas/química , Proteína G de Ligação ao Cálcio S100/química , Cloreto de Sódio/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , SuínosRESUMO
Inorganic phosphate participates in the nonoxidative Cd-dependent dimerization of Cd7-metallothionein (Cd7-MT) and is bound to the MT dimers. In order to obtain insight into the specificity of phosphate-MT interaction, an investigation has been made on the effect of a series of oxyanions and organic phosphates on Cd-induced dimerization of Cd7-MT. It has been demonstrated that from the oxyanions studied, only arsenate and tungstate can promote Cd-induced dimerization of Cd7-MT. Effect of arsenate is quantitatively similar to that of phosphate and appears at submillimolar concentrations, whereas tungstate promotes dimerization of MT at higher, millimolar concentrations. A number of other oxyanions, ie, molybdate, vanadate, and selenate as well as organic phosphates (phenyl phosphate, serine O-phosphate, D-ribose 5-phosphate, L-glycerol 3-phosphate, D-fructose 6-phosphate, D-glucose 6-phosphate, adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), nicotinamideadenine-dinucleotide (NADP), pyridoxal 5-phosphate) had no influence on Cd-induced dimerization of MT. Results obtained strongly indicate a highly specific role of inorganic phosphate in Cd-induced dimerization of Cd7-MT.
Assuntos
Cádmio/farmacologia , Metalotioneína/química , Animais , Ânions/farmacologia , Arseniatos/farmacologia , Biopolímeros , Cromatografia em Gel , Cinética , Fígado/química , Metalotioneína/metabolismo , Organofosfatos/metabolismo , Organofosfatos/farmacologia , Fosfatos/farmacologia , Conformação Proteica , Coelhos , Compostos de Tungstênio/farmacologiaRESUMO
The occurrence of a new monomeric cadmium and phosphate containing metallothionein (MT) form, i.e., Cd13-(Pi)2-MT, is reported. The preferential formation of this MT form from Cd7-MT has been shown to occur in the presence of phosphate and free cadmium at low protein concentration and elevated temperatures. This form displays in gel filtration an apparent molecular weight of 8,900 as opposed to 12,000 for Cd7-MT, suggesting the presence of a more globular structure. This new MT form was characterized by electronic absorption, by circular dichroism (CD), and by 1H, 31P, and 113Cd NMR spectroscopy. The Cd13-(Pi)2-MT form displays at least 24 113Cd signals between 240 and 520 ppm indicating (i) the absence of the original cluster structure of Cd7-MT, (ii) the participation of oxygen and/or nitrogen ligands besides thiolates in metal coordination, and (iii) the presence of more than one stable MT form in the sample. From homonuclear 113Cd COSY and CD studies, evidence for the existence of a cluster structure was obtained. It has been demonstrated that in the Cd13-(Pi)2-MT form two phosphate molecules are bound noncovalently and shown that a very slow exchange with [32P]phosphate in solution (half-life of approximately 56 h) takes place. In the 31P NMR studies, three 31P signals from protein-bound phosphate between 3.5 and 6 ppm have been observed. The 31P signal at 5.7 ppm displayed a heteronuclear 31P-113Cd coupling (J2 = 56 Hz) which provides evidence for direct metal-phosphate coordination. The structural and NMR features of this new MT form are discussed.
Assuntos
Cádmio/análise , Metalotioneína/química , Fosfatos/análise , Animais , Cádmio/farmacologia , Cromatografia em Gel , Dicroísmo Circular , Isótopos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Metalotioneína/isolamento & purificação , Metalotioneína/metabolismo , Fósforo , Conformação Proteica , CoelhosRESUMO
Recently we have demonstrated that the exposure of monomeric Cd7-metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers containing approximately two additional Cd(II) ions/monomer subunit [Palumaa, P., Mackey, E. and Vasák, M. (1992) Biochemistry 31, 2181-2186]. In this study, we demonstrate that inorganic phosphate participates in the Cd-induced dimerization of MT. In the absence of phosphate, Cd-induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an additional peak of MT tetramers were detected in gel-filtration experiments. Arsenate exhibits a similar effect to that of phosphate, whereas a number of other anions, i.e. F-, NO3-, SO4(2-), ClO4-, BO3-, SCN-, HCOO- and CH3COO- had no effect on Cd-induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equilibrium-dialysis experiments using potassium [32P]phosphate established binding of two molecules of phosphate to the dimeric MT form with a dissociation constant, Kd, of 23 +/- 3 microM (20 mM Tris/HCl and 0.1 M KCl, pH 8.0 at 25 degrees C), whereas binding of phosphate was not observed with the monomeric Cd7-MT. The noncovalent nature of phosphate binding to the Cd-induced MT dimers has been demonstrated. The presented data provide the first evidence for the binding of a nonmetallic cellular component to MT.
Assuntos
Cádmio/farmacologia , Fígado/enzimologia , Metalotioneína/biossíntese , Fosfatos/metabolismo , Animais , Cromatografia em Gel , Indução Enzimática , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Metalotioneína/metabolismo , Polímeros , Coelhos , Especificidade por SubstratoRESUMO
The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.
Assuntos
Cádmio/metabolismo , Fígado/enzimologia , Metalotioneína/metabolismo , Animais , Cromatografia em Gel , Dicroísmo Circular , Cinética , Espectroscopia de Ressonância Magnética , Oxirredução , Coelhos , Análise Espectral , UltracentrifugaçãoRESUMO
Effect of temperature on the rate of the bond-breaking step of acetylcholinesterase modification with N,N-dimethylaziridinium ion was studied within 8 to 45 degrees C temperature interval. For this reaction measured by irreversible inhibition of the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine the activation parameters delta H not equal to = 94 kJ/mole and delta S not equal to (25 degrees C) = -9.4 J/mol X deg were obtained. Processing of these data together with our earlier results on spontaneous solvolysis of the aziridinium ion in various water-solvent mixtures showed that all these reactions form a common isokinetic series. That gave evidence of the SN1 mechanism of the alkylation reaction occurring at the acetylcholinesterase active centre. Kinetics of spontaneous decomposition of the covalent bond between the aziridinium reagent and protein molecule was studied. This reaction followed the first-order kinetics and lead to complete liberation of the label from the enzyme, thus suggesting that a single carboxylic or amide group in the active centre was modified by the aziridinium ion.
Assuntos
Acetilcolinesterase , Aziridinas/farmacologia , Azirinas/farmacologia , Inibidores da Colinesterase , Sítios de Ligação , Fenômenos Químicos , Química , Cinética , Especificidade por SubstratoRESUMO
The synthesis of tritium-labeled N,N-demethyl-2-phenylaziridinium has been described. The specific radioactivity of the product obtained was 1,06 TBq/mmole. Kinetics of incorporation of this radioactive label into acetylcholinesterase of cobra venom (Naja naja oxiana) has been studied at 1,05 mM ligand concentration (25 degrees C, pH 7,50. 0,15 M phosphate buffer). Under these conditions two molecules of the radioactive label have been found to react with the enzyme. One molecule incorporates fast with half-life of 4,8 min, not affecting the enzymatic activity. Incorporation of the second label is a slow reaction with half-life of 6 hr and leads to complete inactivation of acetylcholinesterase. Molecular mass of the modified enzyme is 63 +/- 4 kDa and coincides with that of native one.
Assuntos
Aziridinas/síntese química , Azirinas/síntese química , Inibidores da Colinesterase/síntese química , Acetilcolinesterase/metabolismo , Aziridinas/metabolismo , Aziridinas/farmacologia , Sítios de Ligação , Fenômenos Químicos , Química , Inibidores da Colinesterase/metabolismo , Proteínas Neurotóxicas de Elapídeos , Cinética , Peso Molecular , TrítioRESUMO
The kinetics of acetylcholinesterase alkylation with N,N-dimethyl-2-phenylaziridinium ion, the anionic-site-directed affinity label, has been investigated in the presence of alkylboronic acids, which are known as the esteratic-site-directed reversible inhibitors of the enzyme. The ternary complex of the enzyme, the aziridinium ion and alkylboronic acid, are formed in this reaction. In the case of propylboronic acid, for which the complete kinetic analysis of the acceleration effect has been carried out, the 85-fold increase in the rate of the enzyme alkylation reaction has been found. This acceleration effect was connected with the alkylation step, whereas the non-covalent binding of the aziridinium ion in the enzyme active centre was even hindered by the alkylboronic acid. The possible mechanism of this kinetic acceleration phenomenon is discussed with special reference to the kinetic data for the spontaneous solvolysis reaction of the aziridinium ion in water and organic solvents.
Assuntos
Acetilcolinesterase/metabolismo , Aziridinas/metabolismo , Azirinas/metabolismo , Ácidos Borônicos/farmacologia , Animais , Venenos Elapídicos/análise , CinéticaRESUMO
Kinetics of the reaction of N,N-dimethyl-2-phenylaziridinium ions with soluble acetylcholinesterases from cobra venom and electric eel as well as with the membrane-bound acetylcholinesterase and the muscarinic acetylcholine receptor from the cerebral cortex of rat brain was investigated at pH 7,5 and 25 degrees C in 0,15 M phosphate buffer. The inhibition reaction involves the non-covalent binding step followed by the irreversible alkylation step. The spontaneous hydrolysis of the aziridinium compound and the reversible inhibition of the enzymes with the hydrolytic product were taken into account in data treatment. The aziridinium ions were found to bind with similar effectiveness in the active centers of acetylcholinesterases and the muscarinic acetylcholine receptor, however, for the latter the alkylation step is more than 10-fold faster. This difference can be explained by different solvation effects exerted by the active centers of these proteins. The rate constants of the protein alkylation reaction are compared with the kinetic data for decomposition reaction of the aziridinium ion in several solvent-water mixtures, studied separately in the case of five solvents. The basicity of the solvents was found to be the most important factor accelerating the solvolysis.