RESUMEN

We have studied the interaction between lysozyme, destabilized by reducing its -S-S- bonds, and bovine eye lens alpha-crystallin, a member of the alpha-small heat shock protein superfamily. We have used gel filtration, photon correlation spectroscopy, and analytical ultracentrifugation to study the binding of lysozyme by alpha-crystallin at 25 degrees C and 37 degrees C. We can conclude that alpha-crystallin chaperones the destabilized protein in a two-step process. First the destabilized proteins are bound by the alpha-crystallin so that nonspecific aggregation of the destabilized protein is prevented. This complex is unstable, and a reorganization and inter-particle exchange of the peptides result in stable and soluble large particles. alpha-Crystallin does not require activation by temperature for the first step of its chaperone activity as it prevents the formation of nonspecific aggregates at 25 degrees C as well as at 37 degrees C. The reorganization of the peptides, however, gives rise to smaller particles at 37 degrees C than at 25 degrees C. Indirect evidence shows that the association of several alpha-crystallin/substrate protein complexes leads to the formation of very large particles. These are responsible for the increase of the light scattering.

Asunto(s)

Cristalinas/química , Cristalinas/metabolismo , Proteínas de Choque Térmico/química , Muramidasa/química , Animales , Bovinos , Cromatografía en Gel , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Cinética , Luz , Modelos Estadísticos , Péptidos/química , Fotones , Unión Proteica , Transporte de Proteínas , Dispersión de Radiación , Espectrofotometría , Temperatura , Factores de Tiempo , Ultracentrifugación

RESUMEN

Since alpha-crystallin was shown to have in vitro chaperone-like activity, its structure-function relationship has been extensively studied. However, the mechanism of this function is poorly understood. In this study, we monitored the interaction of alpha-crystallin with different proteins namely the insulin B-chain (3.382 kDa), lysozyme (14.4 kDa), and conalbumin (86.18 kDa), all destabilized by dithiothreitol. We found that at 4 degrees C alpha-crystallin prevents the aggregation of destabilized insulin. During the time course of the interaction of alpha-crystallin with the substrate protein, we observed three classes of molecules: the monomeric protein and monomeric alpha-crystallin peptides, alpha-crystallin/substrate protein complexes with a size comparable to alpha-crystallin and large particles. The latter are responsible for the increase of the light scattering of solutions, containing alpha-crystallin/destabilized protein complexes. The molecular exchange between the different populations is temperature dependent and seems to be ruled by the kinetics of the structural changes of the destabilized proteins. At the end all monomeric species are transformed to larger aggregates. The large complexes are enriched in destabilized proteins, compared to the initial ratio alpha-crystallin/substrate protein.

Asunto(s)

Cristalinas/metabolismo , Ditiotreitol/farmacología , Cristalino/metabolismo , Chaperonas Moleculares/química , Animales , Bovinos , Cromatografía en Gel , Chaperonas Moleculares/metabolismo , Unión Proteica/efectos de los fármacos , Espectrofotometría Atómica , Ultracentrifugación

RESUMEN

Lens alphaA- and alphaB-crystallin have been reported to act differently in their protection against nonthermal destabilization of proteins. The nature of this difference, however, is not completely understood. Therefore we used a combination of thermally and solvent-induced structural changes to investigate the difference in the secondary, tertiary and quaternary structures of alphaA- and alphaB-crystallin. We demonstrate the relationship between the changes in the tertiary and quaternary structures for both polypeptides. Far-ultraviolet circular dichroism revealed that the secondary structure of alphaB-crystallin is more stable than that of alphaA-crystallin, and the temperature-induced secondary structure changes of both polypeptides are partially reversible. Tryptophan fluorescence revealed two distinct transitions for both alphaA- and alphaB-crystallin. Compared to alphaB-crystallin, both transitions of alphaA-crystallin occurred at higher temperature. The changes in the hydrophobicity are accompanied by changes in the quaternary structure and are biphasic, as shown by bis-1-anilino-8-naphthalenesulfonate fluorescence and sedimentation velocity. These phenomena explain the difference in the chaperone capacity of alphaA- and alphaB-crystallin carried out at different temperatures. The quaternary structure of alpha-crystallin is more stable than that of alphaA- and alphaB-crystallin. The latter has a strong tendency to dissociate under thermal or solvent destabilization. This phenomenon is related to the difference in subunit organization of alphaA- and alphaB-crystallin where both hydrophobic and ionic interactions are involved. We find that an important subunit rearrangement of alphaA-crystallin takes place once the molecule is destabilized. This subunit rearrangement is a requisite phenomenon for maintaining alpha-crystallin in its globular form and as a stable complex. On the base of our results, we suggest a four-state model describing the folding and dissociation of alphaA- and alphaB-crystallin better than a three-state model [Sun et al. (1999) J. Biol. Chem. 274, 34067-34071].

Asunto(s)

Cristalinas/química , Animales , Bovinos , Dicroismo Circular , Cristalinas/efectos de los fármacos , Cristalinas/aislamiento & purificación , Cristalinas/fisiología , Guanidina/farmacología , Calor , Chaperonas Moleculares/fisiología , Muramidasa/química , Concentración Osmolar , Conformación Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Ultracentrifugación

RESUMEN

The native high molecular mass form of alpha-crystallin, the most important soluble protein in the eye lens, and its low molecular mass form obtained at 37 degrees C in dilute solutions were investigated by synchrotron radiation small-angle X-ray scattering. The alpha-crystallin solutions are polydisperse and good fits to the experimental data can be obtained using distributions of spheres with radii varying between about 5 and 10 nm. In spite of the polydispersity, two different ab initio methods were used to retrieve low resolution shapes from the scattering data. These shapes correspond to the z-average structure of the oligomers. In the absence of any symmetry constraints, the scattering curves of the two forms of alpha-crystallin yield bean-like shapes. The shape corresponding to the low molecular mass form has about 20% less mass at the periphery. Imposing tetrahedral symmetry on the average structures worsens the fit to the experimental data. We emphasized the apparent contradiction between hydrodynamic and molecular properties of alpha-crystallin. An explanation was put forward based on the presence of solvent-exposed flexible C-terminal extensions. We present two bead models ('hollow globule with tentacles' and 'bean with tentacles') based on NMR and cryo-electron microscopy studies and discuss how well they correspond with our data from X-ray scattering, light scattering and analytical ultracentrifugation.

Asunto(s)

Cristalinas/química , Animales , Bovinos , Modelos Moleculares , Dispersión de Radiación , Soluciones , Rayos X

RESUMEN

Alpha-crystallin is the most important soluble protein in the eye lens. It is responsible for creating a high refractive index and is known to be a small heat-shock protein. We have used static and dynamic light scattering to study its quaternary structure as a function of isolation conditions, temperature, time, and concentration. We have used tryptophan fluorescence to study the temperature dependence of the tertiary structure and its reversibility. Gel filtration, analytical ultracentrifugation, polyacrylamide gel electrophoretic analysis, and absorption measurements were used to study the chaperone-like activity of alpha-crystallin in the presence of destabilized lysozyme. We have demonstrated that the molecular mass of the in vivo alpha-crystallin oligomer is about 700 kDa (alpha(native)) while the 550 kDa molecule (alpha(37 degrees C),diluted), which is often found in vitro, is a product of prolonged storage at 37 degrees C of low concentrated alpha-crystallin solutions. We have proven that the molecular mass of the alpha-crystallin oligomer is concentration dependent at 37 degrees C. We have found strong indications that, during chaperoning, the alpha-crystallin oligomer undergoes a drastic rearrangement of its peptides during the process of complex formation with destabilized lysozyme. We propose the hypothesis that all these processes are governed by the phenomenon of subunit exchange, which is well-known to be strongly temperature-dependent.

Asunto(s)

Cristalinas/química , Cristalinas/metabolismo , Animales , Tampones (Química) , Bovinos , Cromatografía en Gel , Cristalinas/aislamiento & purificación , Citoplasma/química , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Cristalino/química , Cristalino/citología , Luz , Chaperonas Moleculares/química , Chaperonas Moleculares/aislamiento & purificación , Chaperonas Moleculares/metabolismo , Peso Molecular , Muramidasa/metabolismo , Fotones , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Dispersión de Radiación , Manejo de Especímenes , Espectrometría de Fluorescencia , Temperatura , Factores de Tiempo , Ultracentrifugación

RESUMEN

We have studied the feasibility of purifying rat C6 glioma plasma membranes by a phase partitioning approach. The purification procedure involves cell homogenization and fractionation with an aqueous two-phase polymer system followed by selective affinity purification of plasma membranes by a wheat germ agglutinin-coupled polymer system. We demonstrate that the two-phase affinity partitioning technique is a simple and efficient method of isolating cell plasma membranes with high purity and yield. Furthermore, the isolated plasma membranes retain their functional integrity, as shown by the high-affinity insulin-like growth factor-I (IGF-I) binding capacity of IGF-I receptors.

Asunto(s)

Fraccionamiento Celular/métodos , Membrana Celular/metabolismo , Glioma/metabolismo , Glioma/ultraestructura , Marcadores de Afinidad , Animales , Centrifugación , Dextranos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Polietilenglicoles , Ratas , Receptor IGF Tipo 1/metabolismo , Células Tumorales Cultivadas , Aglutininas del Germen de Trigo

RESUMEN

We have studied the feasibility of rat C6 glioma cell cultivation on microcarrier beads and the isolation of their plasma membranes from the beads. Cells were cultivated on Cytodex-1 microcarrier beads and the plasma membranes were subsequently isolated from confluent cell monolayers on the beads. This approach yielded approximately 4 x 10(6) cells/ml in a 1 L spinner vessel. Enzymatic assays indicated an 18-fold enrichment of plasma membranes isolated from the beads with minor contamination by other cell organelles. Assay for IGF-I receptor binding capacity revealed that 70% of the total receptor binding capacity could be recovered in the plasma membrane fraction isolated from the beads as compared with the receptor binding capacity of intact cells, demonstrating the functional integrity of the isolated membranes. Electron microscopy and immunofluorescence analysis indicated that the isolated plasma membranes were highly homogeneous with the majority exposing the cytoplasmic surface. Our procedure of C6 glioma cell cultivation on microcarriers and subsequent plasma membrane isolation, provides large quantities of homogeneous and metabolically active membranes which can be used to study receptor-mediated effects on cell proliferation and differentiation.

Asunto(s)

Membrana Celular/química , Glioma/química , Animales , Adhesión Celular , Técnicas de Cultivo de Célula/métodos , División Celular , Membrana Celular/metabolismo , Supervivencia Celular , Dextranos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Glioma/metabolismo , Glioma/patología , L-Lactato Deshidrogenasa/metabolismo , Microscopía Electrónica de Rastreo , Microesferas , Ratas , Receptor IGF Tipo 1/metabolismo , Succinato Deshidrogenasa/metabolismo , Células Tumorales Cultivadas

RESUMEN

The tertiary and quaternary structure of alpha-crystallin is still a matter of controversy. We have characterized the native alpha-crystallin quaternary structure by isolating it at the in vivo temperature and solvent conditions. It can be represented by a distribution of expanded particles with a weight average molar mass of 550,000 g/mol. On decreasing (to 4 degrees C) or increasing (up to 50 degrees C) the temperature, the size distribution increases to larger particles. Only at lower temperatures (4 degrees C), a stable population of particles is obtained with weight average molar mass of 700,000 g/mol. In all conditions, alpha-crystallin behaves as a very expanded particle with a maximum hydrodynamic volume of 3.15 ml/g. The transitions in quaternary structure are rather slow: it takes several hours to evolve from a population of aggregates, characteristic for given solvent conditions, to another distribution in size and quaternary structure on changing the environment. The quaternary structure of alpha-crystallin is an uncharacteristic parameter of the particle: a broad distribution of values can be obtained on changing the environment. Any realistic model should include this property. Our studies favor an open loose structure, where peptides can be added or removed without drastic changes of secondary and tertiary structure of the peptides.

Asunto(s)

Cristalinas/química , Animales , Bovinos , Cristalinas/aislamiento & purificación , Estabilidad de Medicamentos , Luz , Sustancias Macromoleculares , Peso Molecular , Conformación Proteica , Dispersión de Radiación , Solventes , Temperatura

RESUMEN

In the present study, we examined the specific binding of IGF-I and IGF-II to their receptors in C6 glioma cells taken during different growth phases in culture: phase A, early stage of the exponential growth (48 h after seeding); phase B, late stage of the exponential phase (96 h after seeding); phase C, confluent phase (at 144 h after seeding); and phase D, stopped at 48 h post-confluence. Scatchard analysis revealed higher Ka values for the IGF-IR during the exponential phases (A and B). The affinity of IGF-I for its receptor during the post-confluent phase (D) decreased to about half that at phase A (p < 0.01). Although lower at the later phase (D), the number of binding sites of the IGF-IR in the different tested growth stages in culture (A, B, and C) was not statistically different (p > 0.05). Conversely, the number of binding sites of the IGF-II/mannose-6-phosphate receptor appeared to increase during time in culture. The Ka values of the IGF-II/mannose-6-phosphate receptor decreased significantly during the culture time, phase D showing the largest decrease (50%) as compared with phase A (p < 0.005). These binding data suggest that IGF-I and IGF-II receptors are differentially expressed in rat C6 glioma cells in culture and are a function of the growth phase.

Asunto(s)

Ciclo Celular , Glioma/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Animales , Glioma/patología , Ratas , Células Tumorales Cultivadas

RESUMEN

We have studied the quaternary structure of alpha-crystallin in the presence of increasing concentrations of amphiphilic and neutral detergents using gel filtration, light-scattering, boundary and equilibrium sedimentation. We observed a continuous reduction of the molar mass of the polymeric alpha-crystallin on increasing the concentration of sodium dodecyl sulphate from 0.1 mM to 5 mM, ending up with the monomeric peptides. Dodecyltrimethylammonium bromide also disrupts the oligomeric structure of alpha-crystallin but the interaction appears to be cooperative: in the sharp transition region (for a 1 mg/ml protein solution) from 3 to 8 mM of the detergent, only the native protein and a mixture of monomeric and dimeric peptide-DTAB complexes can be observed. Concomitant studies of the circular dichroism in the far UV revealed a substantial decrease of the beta-sheet and increase of the alpha-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent alpha A and alpha B peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the alpha A and alpha B peptides and the formation of the oligomeric alpha-crystallin structure: the binding of the amphiphilic detergent reduces the beta-sheet content, induces the formation of alpha-helix secondary structure and reduces the tendency of the peptide to form large aggregates. The different mechanisms for reducing the oligomeric size by anionic and cationic detergents with identical apolar parts stresses the importance of charge interactions. Our findings support some aspects of the micelle model of alpha-crystallin and can be related to its chaperone activity.

Asunto(s)

Cristalinas/química , Detergentes , Cristalino/química , Conformación Proteica , Animales , Bovinos , Cromatografía en Gel , Dicroismo Circular , Cristalinas/aislamiento & purificación , Glucósidos , Luz , Octoxinol , Dispersión de Radiación , Ultracentrifugación

RESUMEN

The myoglobins of two trematodes, Paramphistomum epiclitum and Isoparorchis hypselobagri, were isolated to homogeneity. The native molecules are monomeric with Mr 16,000-17,000 and pI 6.5-7.5. In each species, at least four different globin isoforms occur. Primary structure was determined at the protein level. The globin chains contain 147 amino acid residues. Although major determinants of the globin fold are conserved, characteristic substitutions are present. A Tyr residue occurs at the helical positions B10 and E7 (distal position). This is confirmed by NMR measurements (Zhang, W., Rashid, K. A., Haque, M., Siddiqi, A. H., Vinogradov, S. N., Moens, L. & La Mar, G. N. (1997) J. Biol. Chem. 272, 3000-3006). A distal Tyr normally provokes oxidation of the iron atom and the inability to bind oxygen, whereas a Tyr-B10 is indicative for a high oxygen affinity. In contrast, trematode myoglobins are functional molecules with a high oxygen affinity. Molecular modeling predicts two possible positions for the aromatic ring of Tyr-E7: one being outside the heme pocket making it freely accessible to the ligand and one within the heme pocket potentially able to form a second hydrogen bond with the iron-bound oxygen. A hydrogen bond between Tyr-B10 and the bound oxygen as in the Ascaris hemoglobin is predicted as well. The predicted structure may explain the high oxygen affinity of the trematode myoglobins.

Asunto(s)

Globinas/química , Mioglobina/química , Paramphistomatidae , Filogenia , Conformación Proteica , Trematodos , Secuencia de Aminoácidos , Animales , Aplysia , Búfalos/parasitología , Simulación por Computador , Fasciola hepatica , Fasciolidae , Globinas/aislamiento & purificación , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Mioglobina/aislamiento & purificación , Paramphistomatidae/aislamiento & purificación , Rumen/parasitología , Homología de Secuencia de Aminoácido , Espectrofotometría , Trematodos/aislamiento & purificación , Vertebrados

RESUMEN

Short range order of the crystallins does account for the transparency of the eye lens. To explain the solution structure of this highly concentrated protein solution on a quantitative basis, the hydrodynamic structure and the interparticle interactions of the proteins have to be known. For that purpose, the light scattering of concentrated solutions of alpha-crystallin has been studied. Starting from the detailed knowledge of the solution parameters of alpha-crystallin in diluted solutions, the structure of concentrated solutions up to 360 mg/ml has been studied using light scattering. Our results indicate that subtle changes in the macromolecular structure such as optical anisotropy or structural asymmetry for part of the alpha-crystallins, which results in solute light-scattering heterogeneity, can dramatically increase the light scattering by the alpha-crystallins and cause solution opacity.

Asunto(s)

Cristalinas/química , Cristalino/química , Animales , Fenómenos Biofísicos , Biofisica , Bovinos , Difusión , Luz , Modelos Biológicos , Dispersión de Radiación , Soluciones , Ultracentrifugación

RESUMEN

We have studied diluted bovine eye lens alpha-crystallin solutions by using light scattering. The protein particles were modeled as hard spheres, showing electrostatic repulsion, due to surplus electric charges, and weak attractive interaction. The repulsive potential VR is defined by the radius of the particles, the Debye length kappa-1, and the number of charges at the Gouy layer; the attractive potential has been described by the London-van der Waals potential and is defined by the Hamaker constant A. We have used the diluted gas approximation and the one component macrofluid model to relate the experimental static factor Ki to the theoretical expression of the interaction potential V(x). This resulted in a Hamaker constant A of 0.06 +/- 0.01 KBT and an effective charge q ranging from 18 +/- 1 at low ionic strength (omega = 0.0022 M) to 50 +/- 5 at high ionic strength (omega = 0.1472 M).

Asunto(s)

Cristalinas/química , Animales , Fenómenos Biofísicos , Biofisica , Bovinos , Difusión , Electroquímica , Técnicas In Vitro , Luz , Sustancias Macromoleculares , Modelos Químicos , Estructura Molecular , Concentración Osmolar , Dispersión de Radiación , Soluciones

Asunto(s)

Péptidos/química , Conformación Proteica , Estructura Terciaria de Proteína

RESUMEN

The application of photon correlation spectroscopy on mammalian eye lenses in vivo is revisited. It is shown that the use of a short wavelength laser type and a logarithmic correlator improves the signal-to-noise ratio to such an extent that shorter measurement times are possible without impairing the information content of the correlation function. Experimental correlation functions obtained in vivo on a rabbit eye lens, are analyzed with several techniques. The histogram approach is most successful for the determination of the distribution function of relaxation processes in the correlation function and proposes four different populations of components in the lens. This result is comparable to that from in vitro measurements on highly concentrated solutions of alpha-crystallins and of fiber cell cytoplasm, the former proteins being the main scattering components both in vivo and in vitro in the eye lens system. Our results indicate that the application of photon correlation spectroscopy on eye lenses in vivo offers new perspectives to use this technique as a fast, noninvasive tool to study relaxation phenomena in normal and cataractous lenses. The sensitivity of the method allows it to be used as an important analytical technique in the study of prevention and treatment of cataract.

Asunto(s)

Cristalino/fisiología , Absorciometría de Fotón , Animales , Rayos Láser , Luz , Matemática , Conejos

RESUMEN

The major protein from the bovine lens fiber cell membranes, the 26-kilodalton protein (major intrinsic protein (MIP26)), has been solubilized in n-octyl-beta-D-glucopyranoside and purified by gel filtration. The final preparation was free of detergent micelles. Gel electrophoresis in denaturing conditions has confirmed the purity of the protein sample. A s20,w of 5.55 S, obtained from analytical ultracentrifugation, and a D20,w of 3.62 x 10(-7) cm2 s-1, obtained from photon correlation spectroscopy, resulted in a molar mass of (176,000 +/- 15,000) g/mol for the protein-detergent complex using the Svedberg relation. The measured detergent content of 0.71 g of detergent/g of protein resulted in a calculated partial specific volume of 0.787 cm3/g for the protein-detergent complex and a molar mass of 103,000 g/mol for the protein moiety. This allowed us to conclude that the protein-detergent complex contains four copies of the MIP26 protein, which supports the suggestion that in vivo the MIP26 molecules cluster in tetramers to form a pore-like structure.

Asunto(s)

Proteínas del Ojo/aislamiento & purificación , Cristalino/análisis , Glicoproteínas de Membrana/aislamiento & purificación , Animales , Acuaporinas , Bovinos , Fraccionamiento Celular , Membrana Celular/análisis , Detergentes , Electroforesis en Gel de Poliacrilamida , Glucósidos , Micelas , Peso Molecular , Análisis Espectral

RESUMEN

The effects of variations in temperature and protein concentration on the renaturation of bovine alpha-crystallin have been examined using gel permeation chromatography, sedimentation analysis, fluorescence spectroscopy and electron microscopy. High protein concentration (3-53 mg/ml) were found to generate heterogenous populations of aggregates. It was concluded that concentrations above 3 mg/ml were inappropriate for renaturation of alpha-crystallin. Aggregates with molecular masses gradually increasing from 461,000 to 695,000 Da were produced with increasing temperature over the range 6-39 degrees C. Electron microscopy demonstrated that the reaggregates were composed predominantly of particles with circular cross-sections and mean diameters of 13-14 nm. As the renaturation temperature increased, increasing amounts of sheet-like structures were observed. Tryptophan accessibility to acrylamide quenching decreased in these aggregates as the size increased. These observations are consistent with the concept that there is no unique quaternary structure, or set of structures, for alpha-crystallin but that the protein can exist in a variety of forms containing different numbers of subunits.

Asunto(s)

Cristalinas/metabolismo , Temperatura , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Feto , Cristalino/análisis , Microscopía Electrónica , Desnaturalización Proteica , Espectrometría de Fluorescencia

RESUMEN

The molecular weight of bovine alpha-crystallin, isolated at 37 degrees C, was studied and found to be about 800,000. This contrasts with the results of Thomson and Augusteyn (Thomson, J. A., and Augusteyn, R. C. (1983) Exp. Eye Res. 37, 367-377) who isolated a species of about half this molecular weight. We show here that this form of alpha-crystallin can only be isolated under unphysiological conditions with regard to buffer pH and ionic strength.

Asunto(s)

Cristalinas/aislamiento & purificación , Corteza del Cristalino/análisis , Cristalino/análisis , Animales , Tampones (Química) , Bovinos , Difusión , Concentración de Iones de Hidrógeno , Peso Molecular , Concentración Osmolar , Temperatura , Ultracentrifugación

RESUMEN

The bovine eye lens protein, alpha L crystallin, has been studied with photon correlation spectroscopy and statical light scattering in the concentration range up to 200 g/l in different solvent conditions. At higher concentration (c greater than 70 g/l) the scattering behavior is quite complicated, which results in nonexponential correlation functions. Three methods have been used for the analysis of these correlation functions, namely, cumulant analysis, sum of two exponentials analysis, and exponential sampling method. These methods resulted in very similar results. The highly concentrated solutions contain two scattering entities: the single alpha L crystallin and a rather heterogeneous population of large clusters. The statical light-scattering experiments can be interpreted in the same way and gave consistent results for the dimensions of the large scattering units. The formation of these clusters, which are strong light scatterers, is superimposed on an increasing degree of correlation between the bulk of the alpha L-crystallins, resulting in a net decrease of light scattering as a function of concentration.

Asunto(s)

Cristalinas/fisiología , Cristalino/fisiología , Animales , Bovinos , Cristalino/ultraestructura , Luz , Modelos Biológicos , Dispersión de Radiación , Soluciones , Análisis Espectral

RESUMEN

The influence of Mg2+ ions on the secondary and tertiary structure of the RNA from bacteriophage MS2 was investigated by small-angle X-ray scattering and light scattering and by sedimentation experiments. The analysis of the outer part of the X-ray scattering curve obtained at low temperature in the absence of Mg2+ yielded a cross-section radius of gyration of 0.88 nm and a mass per unit length of 1720 g mol-1 nm-1. Very similar values for these parameters, which refer to the secondary structure of the RNA molecule, were also derived from the X-ray scattering curves obtained in the presence of different amounts of Mg2+ (0.07 to 1 ions per nucleotide). On the contrary, the inner part of the X-ray scattering curves turned out to be highly dependent on the Mg2+ concentration: the cross-section radius of gyration and the mass per unit length, which were determined from the scattering curves at small angles as parameters related to the tertiary structure of the RNA, amounted to 3.11 nm and 4000 g mol-1 nm-1, respectively, in the absence of Mg2+ and increased significantly upon raising the concentration of Mg2+. The increase of these structural parameters was found to be accompanied by a decrease of the overall radius of gyration (as revealed indirectly by X-ray scattering and directly by light scattering measurements) and by an increase of the sedimentation coefficient. The results from the investigations of the RNA at low temperature clearly establish the existence of double-stranded structures down to very low Mg2+ concentrations as well as the occurrence of Mg2+ induced changes of the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Bacteriófagos/metabolismo , Magnesio/farmacología , ARN Viral/análisis , Luz , Conformación de Ácido Nucleico , ARN Viral/aislamiento & purificación , Dispersión de Radiación , Temperatura , Ultracentrifugación , Difracción de Rayos X