RESUMEN
A section-selective three-dimensional phosphorus-31 chemical shift imaging (CSI) experiment was evaluated as the spatial localization method for spectroscopy in an integrated clinical magnetic resonance (MR) imaging and spectroscopy examination. The results of a CSI experiment can be displayed as either spectra related to specific voxels or "metabolite maps," in which the relative concentration of a given metabolite is displayed as an overlay of the MR image. This method was applied to the study of a soft-tissue mass and to a meningioma. The total imaging time in each case was 17 minutes with a voxel size of 27 cm3 in the extremity and 64 cm3 in the brain. The total time to set up this part of the study was about 10 minutes. No additional shimming was necessary when the center of the field of view selected for the CSI experiment was located at or near isocenter. The promising results obtained with this approach make the CSI method an attractive choice of spatial localization method.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Neoplasias Meníngeas/diagnóstico , Meningioma/diagnóstico , Neoplasias de los Tejidos Blandos/diagnóstico , Adulto , Anciano , Femenino , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Modelos Estructurales , FósforoRESUMEN
Different methods of preparing rabbit liver 113Cd,ZnMT in vitro were investigated in an attempt to reproduce the distinctive and reproducible manner in which the metals are distributed between the 2 clusters in vivo in protein produced in response to Cd administration. 113Cd NMR was used to demonstrate that the native metal distribution is not produced by Cd displacement of Zn from ZnMT, but rather by a direct metal interchange reaction between CdMT and ZnMT. Kinetic studies of this heretofore unknown reaction indicate that even at low physiological concentrations, CdMT and ZnMT cannot coexist without rapidly exchanging metals to produce mixed-metal protein species containing Cd and Zn located preferentially in the 4- and 3-metal clusters, respectively. The self-exchange reaction of Cd among the 7 binding sites of CdMT was also investigated and it was found that exchange of Cd among sites in the 3-metal cluster was very rapid (time-scale of seconds) while among sites of the 4-metal cluster or between clusters it was several orders of magnitude slower.
Asunto(s)
Cadmio/metabolismo , Metalotioneína/metabolismo , Zinc/metabolismo , Animales , Cinética , Hígado/análisis , Espectroscopía de Resonancia Magnética , ConejosRESUMEN
Azurin, a blue copper protein from Pseudomonas aeruginosa, and several derivatives of azurin have been studied by differential scanning calorimetry. Two well-separated, irreversible transitions are observed in a scan of apoazurin under a variety of conditions, and they are assigned to distinct steps in the denaturation process. No specific structural component can be assigned to the lower temperature transition, but a "flap" structure which is found near the metal binding site may be involved. Circular dichroic spectra suggest that melting of the beta-sheet structure, the main structural motif in the native protein, occurs during the second transition. With the exceptions of the Ni(II) and p-(hydroxymercuri)benzoate derivatives, the transitions are superposed in the metalated forms, and the enthalpies of denaturation are more endothermic. By comparison with other first-row divalent transition ions and especially Zn(II), the Cu(II) derivative exhibits the most endothermic denaturation process. Along with other data, this suggests that the binding energy is greater for Cu(II). It is postulated that the selectivity for copper over zinc arises because of the irregular binding geometry offered by the folded protein. Denaturation of the Hg(II) derivative is even more endothermic, confirming that the type 1 binding site has a very great affinity for Hg(II). Finally, when substoichiometric amounts of Hg(II) are added to the apoprotein, there is evidence that a novel mercury-bridged dimer of azurin forms.
Asunto(s)
Azurina/metabolismo , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Azurina/análogos & derivados , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Concentración de Iones de Hidrógeno , Plantas/metabolismo , Plastocianina/metabolismo , Conformación Proteica , Desnaturalización Proteica , Pseudomonas aeruginosa/metabolismoRESUMEN
113Cd NMR analysis of rabbit liver metallothionein 2 reconstituted with 113Cd at all seven binding sites has previously indicated that the metals are arranged in two metal-thiolate clusters [Otvos, J.D., & Armitage, I.M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. Spectra of the protein always contained more than seven resonances, however, suggesting the samples were in some way heterogeneous. Results of a recent study of 113Cd metallothionein reconstituted in a different manner but also giving spectra with more than seven resonances have been interpreted as arguing against the two-cluster model of metal binding and in favor of a model in which structural flexibility of the protein allows many configurational substates of the cluster(s) to coexist [Vasak, M., Hawkes, G.E., Nicholson, J.K., & Sadler, P.J. (1985) Biochemistry 24, 740-747]. Data are presented here that indicate that dimers and larger oligomers of metallothionein formed as byproducts of metal reconstitution are the likely source of at least some of the 113Cd resonances attributed by these workers to configurational substrates. Removal of the contaminating oligomers by gel filtration yields a verifiably homogeneous protein whose 113Cd spectrum consists of seven resonances of comparable intensity. Unambiguous confirmation of the existence and structures of the two previously proposed metal-thiolate clusters was obtained by two-dimensional chemical shift correlation spectroscopy and spectral simulation of the 113Cd-113Cd splitting patterns of the individual resonances.
Asunto(s)
Hígado/metabolismo , Metalotioneína/metabolismo , Animales , Sitios de Unión , Cadmio , Isótopos , Espectroscopía de Resonancia Magnética/métodos , Metalotioneína/aislamiento & purificación , Unión Proteica , ConejosRESUMEN
Hepatic metallothionein (MT) isolated from Cd-exposed animals always contains Zn (2-3 mol/mol of protein) in addition to Cd (4-5 mol/mol of protein), and the two metals are distributed in a nonuniform, but reproducible, manner among the seven binding sites of the protein's two metal-thiolate clusters. Different methodologies of preparing rabbit liver Cd, Zn-MT in vitro were investigated to provide insight into why such a distinct mixture of mixed-metal clusters is produced in vivo and by what mechanism they form. 113Cd NMR spectra of the products of stepwise displacement of Zn2+ from Zn7-MT by 113Cd2+ show that Cd binding to the clusters is not cooperative (i.e., clusters containing exclusively Cd are not formed in preference to mixed-metal Cd, Zn clusters), there is no selective occupancy of one cluster before the other, and many clusters are produced with a nonnative metal distribution indicating that this pathway is probably not followed in vivo. In contrast, the surprising discovery was made that the native cluster compositions and their relative concentrations could be reproduced exactly by simply mixing together the appropriate amounts of Cd7-MT and Zn7-MT and allowing intermolecular metal exchange to occur. This heretofore unknown metal interchange reaction occurs readily, and the driving force appears to be the relative thermodynamic instability of three-metal clusters containing Cd. With this new insight into how Cd,Zn-MT is likely to be formed in vivo we are able for the first time to postulate rational explanations for previous observations regarding the response of hepatic Zn and metallothionein levels to Cd administration.
Asunto(s)
Metalotioneína/metabolismo , Animales , Sitios de Unión , Cadmio , Isótopos , Hígado/metabolismo , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , ConejosRESUMEN
Which cysteine of apostellacyanine reacts with 4-vinylpyridine in 6 M guanidine-HC1 depends upon the pH. We infer that a disulfide switch occurs at higher pH and that in the native protein the disulfide bridge occurs between Cys-59 and Cys-93. Thus Cys-87, which is homologous to the cysteine ligands of azurin and plastocyanin, is available to bind copper.
Asunto(s)
Disulfuros/análisis , Metaloproteínas/análisis , Proteínas de Plantas/análisis , Secuencia de Aminoácidos , Cisteína/análisisRESUMEN
The 113Cd NMR spectra of plastocyanin (Spinacea), stellacyanin (Rhus vernicifera), and two azurins (Pseudomonas aeruginosa and Alcaligenes faecalis) have been measured after introducing Cd(II) into the blue copper-binding sites. Relative to Cd(C1O4)2 the chemical shifts are 432, 380, 372, and 379 ppm, respectively, all of which are found to be reasonable values for binding sites containing a cysteine thiolate ligand. The 113Cd resonances of the cadmium derivatives of stellacyanin and the azurins are so near the same that the proteins must present very similar metal-binding sites. In contrast the plastocyanin derivative resonates about 50 ppm further downfield which may signal a change in coordination number. The spin lattice relaxation times of the 113Cd resonances are of the order of 0.1 s, and a major portion of the relaxation apparently occurs through the chemical shift anisotropy mechanism. At 13 degrees C the 113Cd resonance of Psuedomonas azurin shifts slightly downfield with increasing pH. This is explained by a small change in the environment about cadmium which occurs as a result of the conformational change that attends the titration of His-35.