RESUMEN
A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed.
Asunto(s)
Bacteriorodopsinas/química , Mioglobina/química , Agua/química , Conformación Proteica , Temperatura , TermodinámicaRESUMEN
The reactivities of the sulfhydryl groups of rat, turkey, human, and calf hemoglobin were studied together with the enzyme activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutaredoxin in lysed erythrocytes to evaluate their roles in regulating redox homeostasis. The results of -SH reactivity showed rate constants spanning four orders of magnitude (k2, calf, 6.67 M-1 s-1; rat -SH fast reacting, 2.8 x 10(4) M-1 s-1). Enzyme activities of glucose-6-phosphate dehydrogenase ranged from 0.402 U/ml (calf) to 0.900 U/ml (rat), glutathione reductase from 0. 162 U/ml (rat) to 0.381 U/ml (human), glutaredoxin from 0.778 U/ml (rat) to 2.28 U/ml (turkey), and glutathione peroxidase from 2.07 U/ml (human) to 27.3 U/ml (rat). Blood samples of the four species were also treated with 0.5-1.5 mM tert-butyl hydroperoxide (t-BOOH) or diamide, and levels of glutathione-derived species [GSH, GSSG, and glutathione-protein mixed disulfides (GS-SP)] were determined within 120 min and related to the corresponding protein -SH group (PSH) reactivities and enzyme repertoires. In all cases t-BOOH rapidly transformed GSH into GSSG by the action of glutathione peroxidase; GSSG was in turn transformed into GS-SP, according to the reaction GSSG + PSH --> GS-SP + GSH, or reduced back to GSH by glutathione reductase. The GSSG reduction was more efficient in rat and human blood, due to the contribution of the fast-reacting -SH of hemoglobin, in the rat, and to the efficiency of the enzyme repertoire of human blood. Calf blood showed a relatively low capacity to restore normal values after oxidative stress, due to its low PSH reactivity and the weak contribution of its enzymes. Diamide treatment, which is known to react nonenzymatically with thiols, gave increased GS-SP levels in rat and turkey, but not in human and calf blood, as expected from the different corresponding PSH reactivities. Species with relatively high PSH reactivity and glucose 6-phosphate dehydrogenase activity, such as the rat, therefore had a higher antioxidant capacity than species (calf) in which these parameters were relatively low.
Asunto(s)
Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Homeostasis , Oxidorreductasas , Compuestos de Sulfhidrilo/sangre , Animales , Bovinos , Diamida/farmacología , Activación Enzimática/efectos de los fármacos , Eritrocitos/enzimología , Eritrocitos/fisiología , Glucosafosfato Deshidrogenasa/metabolismo , Glutarredoxinas , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Hematócrito , Hemoglobinas/fisiología , Humanos , Cinética , Masculino , Modelos Biológicos , Modelos Químicos , Oxidación-Reducción , Peróxidos/farmacología , Proteínas/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno , Compuestos de Sulfhidrilo/fisiología , Factores de Tiempo , Pavos , terc-ButilhidroperóxidoRESUMEN
Advances in experimental and computational methodologies have led to a recent renewed interest in the Hofmeister series and its molecular origins. New results are surveyed and assessed. Insights into the underlying mechanisms have been gained, although deeper molecular understanding still seems to be elusive. The principal reason appears to be that the Hofmeister series emerges from a combination of a general effect of cosolutes (salts, etc.) on solvent structure, and of specific interactions between the cosolutes and the solute (protein or other biopolymer). Hence every system needs to be studied individually in detail, a state of affairs which is likely to continue for some time. A deeper understanding of the Hofmeister series can be an extraordinarily valuable guide to designing experiments, including not only those probing the series per se, but also those designed to elucidate the adsorption, aggregation and stabilization phenomena which underlie so many biological events. The aim of this review is to provide an up-to-date framework to guide such understanding, consolidating recent advances in the many fields on which the Hofmeister series impinges.
Asunto(s)
Proteínas/aislamiento & purificación , Fenómenos Biofísicos , Biofisica , Cristalización , Estabilidad de Medicamentos , Proteínas/química , Sales (Química) , Solubilidad , Solventes , Propiedades de Superficie , TermodinámicaRESUMEN
The male reproductive system of the mollusc bivalve Unio elongatulus contains two distinct forms of alpha-L-fucosidase, one present in the gonad fluid and a second one associated with the sperm plasma membrane. Both activities were purified to homogeneity. The soluble seminal plasma enzyme had an oligomeric MW of 56 kDa as determined by MALDI-TOF mass spectrometry, whereas the enzyme purified from sperm plasma membranes had an MW of 68 kDa. Analyzed by lectin blotting with ConA and PNA, the 68 kDa enzyme did not bind either lectin, whereas the 56 kDa form bound ConA only. Both fucosidases followed a Michaelis-Menten kinetics with the K(m) of the sperm-bound enzyme being 7.1 x 10(-4) M and that of the seminal enzyme being 9.1 x 10(-4) M. Both had a pH optimum of 5.0.
Asunto(s)
Espermatozoides/enzimología , alfa-L-Fucosidasa/metabolismo , Animales , Membrana Celular/enzimología , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Lectinas/metabolismo , Masculino , Peso Molecular , Moluscos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Semen/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , alfa-L-Fucosidasa/química , alfa-L-Fucosidasa/aislamiento & purificaciónRESUMEN
The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library. Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively. The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins. A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.
Asunto(s)
Hígado/enzimología , Tiosulfato Azufretransferasa/genética , Glándulas Suprarrenales/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Pollos , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Escherichia coli/genética , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Homología de Secuencia de Ácido NucleicoRESUMEN
Tetranitromethane, C(NO2)4, a reagent for tyrosyl residues, was found to inactivate irreversibly rabbit skeletal muscle glycogen phosphorylase b. Under the chosen conditions seven tyrosyl residues, namely Tyr-75, 203, 262, 280, 403, 552 and 647, were found to be nitrated. Inactivation was prevented by the presence of the allosteric activator 5'-AMP during nitration. Under these latter conditions one of the reactive tyrosyl residues was not modified by C(NO2)4; thus, this residue appeared to be essential for either catalytic activity or allosteric activation. Tryptic digests of phosphorylase b, reacted with C(NO2)4 in the absence and presence of 5'AMP, were fractionated by gel filtration. The peptide mixtures were further purified by reverse-phase HPLC. One of the peptides contained the tyrosyl residue which was modified by C(NO2)4 only in the absence of 5'AMP. The sequence of this peptide was determined. The amino acid residue which is responsible for the loss of activity upon reaction with C(NO2)4 was identified in the amino acid sequence of phosphorylase b as tyrosine-75. Of the other residues modified in the presence and in the absence of C(NO2)4, tyrosine-403 contributes to the glycogen-storage site whereas Tyr-280 is close to the alpha-D-glucose-binding site. These residues, exposed to the solvent both in the presence and in the absence of 5'AMP, are not essential for catalytic activity.
Asunto(s)
Metano/análogos & derivados , Fosforilasa b/antagonistas & inhibidores , Fosforilasas/antagonistas & inhibidores , Tetranitrometano/farmacología , Tirosina/análisis , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Catálisis , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Músculos/enzimología , Fragmentos de Péptidos/análisis , ConejosRESUMEN
Sulphydryl groups of E. coli tryptophanase (L-tryptophan indole lyase, E.C. 4.1.99.1) were made to react with a fluorescent maleimide derivative, N-(4-anilino-1-naphthyl)maleimide(ANM). By carefully controlling the reaction conditions it was possible to limit the extent of sulphydryl group modification. The modified enzyme was digested with (L-1-tosylamide-2-phenylethyl chloromethyl ketone)-trypsin. The fluorescent peptides obtained were analysed by reversed-phase high-performance liquid chromatography on a C18 column with a dual-monitoring system consisting of a UV and a fluorescence monitor connected in tandem. This was followed by the determination of the amino acid composition of the fluorescent peptides. Comparison of these results with the known, complete primary structure of tryptophanase from the K-12 strain of E. coli allowed the assignment of position 298 to the cysteine residue, which is more selectively modified by ANM under the conditions chosen and is involved in the maintenance of the catalytic activity.
Asunto(s)
Liasas/análisis , Compuestos de Sulfhidrilo/análisis , Triptofanasa/análisis , Aminoácidos/análisis , Cromatografía Líquida de Alta Presión , Colorantes Fluorescentes , Hidrólisis , Espectrofotometría Ultravioleta , TripsinaAsunto(s)
Acetilglucosaminidasa/fisiología , Hexosaminidasas/fisiología , Óvulo/enzimología , Erizos de Mar/fisiología , Espermatozoides/enzimología , Acetilglucosaminidasa/metabolismo , Animales , Detergentes/farmacología , Femenino , Masculino , Peso Molecular , Óvulo/fisiología , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Espermatozoides/fisiología , TemperaturaRESUMEN
We have purified a beta-N-acetylglucosaminidase from the hepatopancreas of the octopus which we have called beta I. The enzyme was homogeneous as judged by Sephadex column chromatography, isoelectric focusing, non-denaturing gel electrophoresis at two different pH and with sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The native protein has an apparent molecular weight of 120 000 and we can conclude that it is a tetramer made up of two alpha and two beta subunits with apparent Mr of 27 000 and 34 000, respectively. Using NMR spectroscopy we have examined the specificity of beta I and have established that the enzyme hydrolyses the beta 1,4 linkage of N-acetylglucosamine but at only a specific site of the substrates used, two glycopeptides isolated from ovalbumin. To our knowledge this is the first known exoglycosidase which has both linkage and site specificity.
Asunto(s)
Acetilglucosaminidasa/aislamiento & purificación , Hexosaminidasas/aislamiento & purificación , Octopodiformes/enzimología , Animales , Fenómenos Químicos , Química , Focalización Isoeléctrica , Espectroscopía de Resonancia Magnética , Peso Molecular , Especificidad por SustratoRESUMEN
A protein methylase from calf-lifer nuclei was partially purified by sonication of the nuclear pellet at high ionic strength, chromatin removal and ammonium sulphate fractionation of the solubilized activity.
Asunto(s)
Hígado/enzimología , Proteína Metiltransferasas/metabolismo , Animales , Bovinos , Núcleo Celular/enzimología , Activación Enzimática , Concentración de Iones de Hidrógeno , Cinética , S-Adenosilmetionina/metabolismo , Albúmina Sérica Bovina/metabolismoRESUMEN
The efficiency of transcription of both mammalian and bacterial templates by 3 distinct multiple forms of DNA-dependent RNA polymerase from calf liver nuclei has been determined. The homologous template and salmon sperm DNA were more efficiently transcribed than bacterial templates. The native DNA/denatured DNA activity ratio of the 2 alpha-amanitin-insensitive forms was found consistent with that described for enzymes exhibiting nucleolar localization.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Hígado/enzimología , Animales , Bovinos , Núcleo Celular/enzimología , Desnaturalización de Ácido Nucleico , Especificidad por Sustrato , Moldes GenéticosRESUMEN
Large scale purification and preparation of calf liver nuclei was accomplished by high speed centrifugation of a fraction enriched in nuclei ('nuclear homogenate') through 1.8 M sucrose by means of a Beckman CF-32 Ti continuous flow rotor. In comparison with methods involving the use of conventional high capacity rotors, larger volumes of homogenate could be processed. This method was used to prepare nuclei from calf liver for the preparation of DNA-dependent RNA polymerases. The use of continuous flow ultracentrifugation avoids time-consuming manipulations, thus allowing handling of large quantities of tissue.
Asunto(s)
Núcleo Celular/ultraestructura , Hígado/ultraestructura , Animales , Bovinos , Fraccionamiento Celular/métodos , Núcleo Celular/enzimología , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Hígado/enzimología , UltracentrifugaciónRESUMEN
An electrophoretically homogeneous preparation of endo-polygalacturonase (poly(1,4-alpha-D-galacturonide)glycanohydrolase, EC 3.2.1.15) from culture filtrates of Rhizoctonia fragariae, a pathogenic agent in strawberry plants, was resolved into two isoenzymes when subjected to isoelectrofocusing in a narrow pH range. The isoelectric points of the two isoenzymes were 6.76 +/- 0.03 and 7.08 +/- 0.05. The two polygalacturonases exhibited similar substrate specificity, pH optimum and pattern of degradation of sodium polypectate. The two enzymes consisted of a single polypeptide chain which had an apparent molecular weight of 36 000 as determined by gel filtration on Sephadex G-100.
Asunto(s)
Glicósido Hidrolasas , Isoenzimas , Hongos Mitospóricos/enzimología , Poligalacturonasa , Rhizoctonia/enzimología , Glicósido Hidrolasas/metabolismo , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Peso Molecular , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismoAsunto(s)
Metano , Fosforilasas , Tetranitrometano , Adenosina Monofosfato/farmacología , Regulación Alostérica , Sitio Alostérico/efectos de los fármacos , Animales , Sitios de Unión/efectos de los fármacos , Fenómenos Químicos , Química , Metano/análogos & derivados , Músculos/enzimología , Fosforilasas/antagonistas & inhibidores , Conformación Proteica/efectos de los fármacos , Conejos , Tetranitrometano/antagonistas & inhibidores , Tetranitrometano/farmacología , Tirosina , UltracentrifugaciónRESUMEN
A DNA-dependent RNA polymerase has been isolated from Caldariella acidophila, a thermophilic bacterium living in acidic hot springs at temperatures ranging from 63 to 89 degrees C. The enzyme was purified 180-fold and is composed of five different subunits having the following molecular weights: a = 127000, b = 120000, c = 72000, d = 65000, and e = 38000. The enzyme is activated by Mn2+ and Mg2+ and exhibits optimal activity in the presence of 0.5 mM Mn2+. The activity depends on ionic strength, with a maximum at 0.25 M KCl, and exhibits a pH optimum at 7.8 in the presence of Tris-HCl buffer. The enzyme shows a high degree of thermophilicity, its temperature optimum being 80 degrees C in the in vitro assay. The thermophilicity of C. acidophila RNA polymerase allows studies on enzyme-template interactions to be performed in a temperature range where many templates are close to their Tm.