RESUMEN
Parvalbumins (Pv) are calcium-binding proteins present mainly in the muscle and nervous system where they act as a Ca(2+) buffer. Our previous work demonstrated the presence of Pv-I in carp semen and indicated the presence of a second Pv (Pv-II). The purpose of the present work was to identify, purify and determine the full-length cDNA sequence of Pv-II from carp testis. Pv-II from seminal plasma was purified by ion-exchange chromatography (IEC) and preparative electrophoresis, while the Pv-II from spermatozoa was purified by IEC, gel filtration and preparative electrophoresis. The purified Pv-II was submitted to an analysis of molecular mass, isoelectric point (pI), amino-acid sequence and oligomerisation ability. The amino-acid sequence was used to construct primers and obtain the full-length cDNA sequence of seminal-specific Pv-II from carp testis. Analysis of the cDNA sequence indicated that carp-testis Pv-II was distinct from carp-muscle parvalbumins. Pv-II was distinct from Pv-I regarding sequence, molecular mass and pI. Both parvalbumins had the ability to form oligomers or to bind to other proteins. Carp seminal plasma had a protective effect against parvalbumin oligomerisation. Pv-II underwent post-translational modification such as n-acetylation and cysteinylation. The present study is the first to report the full-length cDNA sequence of parvalbumin from carp testis.
Asunto(s)
Carpas/genética , ADN Complementario/genética , Parvalbúminas/genética , Parvalbúminas/aislamiento & purificación , Semen/química , Análisis de Secuencia de ADN , Espermatozoides/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carpas/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Punto Isoeléctrico , Masculino , Datos de Secuencia Molecular , Peso Molecular , Parvalbúminas/química , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ProteínaRESUMEN
Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone (2)J(NC') couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental (2)J(NC') couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide. Findings indicate that elevated (2)J(NC') couplings may serve as reporters of structural strain in the protein backbone imposed by protein folds. Such information, supplemented with the H-bond strengths derived from (h3)J(NC') couplings, provides useful insight into the overall energy profile of the protein backbone in solution.
Asunto(s)
Pliegue de Proteína , Proteínas/química , Proteínas Bacterianas/química , Calmodulina/química , Isótopos de Carbono , Proteínas de Unión a Ácidos Grasos/química , Humanos , Enlace de Hidrógeno , Conformación Molecular , Isótopos de Nitrógeno , Oligopéptidos/química , Parvalbúminas/química , Proteínas de Plantas/química , Conformación Proteica , Ubiquitina/químicaRESUMEN
Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.
Asunto(s)
Canales de Calcio Tipo N/química , Calcio/química , Regulación de la Expresión Génica , Animales , Western Blotting , Tampones (Química) , Calbindinas , Calcio/metabolismo , Canales de Calcio Tipo N/metabolismo , Línea Celular , Citoplasma/metabolismo , ADN Complementario/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Ácido Egtácico/farmacología , Electrofisiología , Humanos , Cinética , Modelos Biológicos , Parvalbúminas/química , Técnicas de Placa-Clamp , Ratas , Proteína G de Unión al Calcio S100/química , TransfecciónRESUMEN
In the course of the study of structure-functional properties and molecular mechanisms of neuropeptides and of low molecular weight proteins of the central nervous system we succeeded in isolating from the soluble fraction of bovine hypothalamus a protein having M(r) 11897.3, according to mass spectral analysis. The purification procedure was mainly based on reversed phase HPLC. As the N-terminus of the molecule was found to be blocked, we have subjected it to CNBr degradation. By Edman microsequence analysis of the peptide fragments and by data base searching the isolated substance was identified as parvalbumin alpha (PRVA)-one of the calcium-binding proteins. However, its primary structure was found not to be identical to that of the known PRVAs from other sources. One of the features of PRVA is its stability. Being subjected to an exhausting purification procedure it retains its complete structure. As neuropeptides and low molecular weight proteins are found to be polyfunctional, a central question concerns the biological role of PRVAs in terms of "where and when" they express their action.
Asunto(s)
Proteínas de Unión al Calcio/química , Hipotálamo/química , Proteínas del Tejido Nervioso/química , Parvalbúminas/química , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/fisiología , Bovinos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/fisiología , Parvalbúminas/fisiología , Relación Estructura-ActividadRESUMEN
Rat parvalbumin (PV) and oncomodulin (OM) differ in the affinity and selectivity of metal binding to their CD site, which is a high-affinity Ca2+/Mg(2+)-mixed site in PV and a low-affinity Ca(2+)-specific site in OM. To assess to what degree the Ca2+/Mg2+ specificity and affinity of an EF-hand motif in a protein is intrinsically determined by its sequence, the complete CD sites were exchanged, yielding two chimeras, [S41-Q71]PV and [D41-S71]OM. The optical characteristics of a Trp102, inserted in the hydrophobic core of PV, OM and the two chimeras, are very similar in all four proteins, which suggests that the hydrophobic core is qualitatively similar in the chimeras as in the parent proteins. Direct Ca2+ and Mg2+ binding monitored by flow dialysis and gel filtration revealed that [S41-Q71]PV binds only one Mg2+ with an intrinsic affinity K'Mg2+ of 3.0 x 10(4) M-1 and two Ca2+ with an identical K'Ca2+ of 4.4 x 10(6) M-1, whereas [D41-S71]OM binds two Mg2+ with a mean K'Mg2+ of 2 x 10(4) M-1 and two Ca2+ with a K'Ca2+ of 1.3 x 10(7) M-1. K'Ca2+ of the CD site of [S41-Q71]PV was 2.5-fold higher than of the CD site in [W102]OM, but 5-6-fold lower than that of the CD site in [W102]PV. In [D41-S71]OM, K'Ca2+ of the CD site was twofold lower than in [W102]PV, but eightfold higher than in [W102]OM. These results indicate that the sequence of the CD site determines its Ca2+/Mg(2+)-specificity, whereas its affinity for Ca2+ influenced by the protein into which the CD site is inserted. The inserted CD site in turn influences the affinity of the EF site to which it is paired in the host protein and the paired sites display an equalized affinity for Ca2+. Mg2+ decreases the affinity of the chimeras for Ca2+, but not according to a simple competition model. The Mg2+ antagonism is much more pronounced in [D41-S71]OM than in [S41-Q71]PV, but in each chimera the CD and EF site are quantitatively affected in the same manner. Thus, [S41-Q71]PV which can only bind a single Mg2+ ion, displays a Ca2+/Mg(2+)-antagonism for both sites with a KMg.compet of 2.3 x 10(2) M-1. These results confirm the 'equalizer' principle in the cation-binding parameters of [S41-Q71]PV: both sites display the same Ca2+ affinity and Mg2+ antagonism. In [D41-S71]OM with its two Ca2+/Mg2+ sites the antagonism shows qualitatively the same complexity as in wild-type PV, although it is somewhat weaker in amplitude.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Magnesio/metabolismo , Parvalbúminas/química , Parvalbúminas/metabolismo , Animales , Sitios de Unión , ADN Complementario , Cinética , Espectrometría de Masas , Metales/análisis , Mutagénesis Sitio-Dirigida , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Especificidad por Sustrato , Compuestos de Sulfhidrilo/análisisAsunto(s)
Proteínas de Unión al Calcio/fisiología , Genes , Parvalbúminas/fisiología , Animales , Sitios de Unión , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Células Cultivadas , ADN Complementario/genética , Regulación de la Expresión Génica , Genes de Partícula A Intracisternal , Secuencias Hélice-Asa-Hélice , Humanos , Magnesio/metabolismo , Ratones , Ratones Transgénicos , Modelos Moleculares , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Relajación Muscular/fisiología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Parvalbúminas/química , Parvalbúminas/genética , Conformación Proteica , Ratas , Relación Estructura-Actividad , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
Previously a rat parvalbumin mutant protein PVF102W was constructed with a reporter Trp at position 102 in the middle of the hydrophobic center [Pauls, T. L., et al. (1993) J. Biol. Chem. 268, 20897-20903]. In the present study three new parvalbumin mutant proteins, derived from PVF102W and containing alterations at positions essential for Ca2+ binding in either one of the two Ca(2+)-binding sites (PV-CD and PV-EF) or in both (PV-CD/-EF), were expressed and purified. With the flow dialysis method it was established that both PV-CD and PV-EF bind 1 Ca2+ with affinity constants KCa of 1.1 x 10(7) and 3.2 x 10(6) M-1, respectively. Mg2+ binding, monitored by equilibrium gel filtration in the absence of Ca2+, showed that both mutants bind 1 Mg2+ with KMg = 8 10(4) for PV-CD and 3 x 10(3) M-1 for PV-EF. Compared to the parameters of the parent mutant PVF102W (two sites with equal affinities of 2.7 x 10(7) and 3 x 10(4) M-1 for Ca2+ and Mg2+, respectively), these data indicate that inactivation of the EF site, much more than of the CD site, impairs divalent cation binding. The binding of Ca2+ and Mg2+ is mutually exclusive, indicative of a Ca2+/Mg2+ mixed site. However, as for PVF102W, the KMg values obtained from the competition equation are approximately 40-fold lower than the affinities measured by direct binding. PV-CD/-EF binds neither Ca2+ nor Mg2+. Trp fluorimetry revealed that in the three mutant PVs the residue Trp-102 is deeply buried in the hydrophobic core.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Parvalbúminas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , ADN Complementario/genética , Técnicas In Vitro , Magnesio/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Parvalbúminas/química , Parvalbúminas/genética , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
Comparison of the immunocytochemical localizations revealed distinct patterns of differential distribution and overlapping of calbindin-D28K (CB-D28K), calretinin (CR), calmodulin (CM) and parvalbumin (PV) in the rat spinal cord. In some areas, one of the four calcium-binding proteins (CBPs) appears to be predominant, for example, CB-D28K in lamina I and ependymal cells, PV at the inner part of laminae II, CR in laminae V and VI and CM in motoneurons of lamina IX. In other regions of the spinal cord, more than one CBPs was abundant. CB-D28K and CR were similarly distributed in lamina II and the lateral spinal and cervical nucleus; CM and PV were similarly abundant in the ventromedial dorsal horn, internal basilar and central cervical nucleus; CR and PV were similarly abundant in the ventromedial dorsal horn, internal basilar and central cervical nucleus; CR and PV were similarly heterogeneous in the gracile fasciculus from caudal to rostral spinal cord. In the sacral dorsal gray commissure, the distribution patterns of CR and PV were clearly complementary. The unilateral ganglionectomies resulted in a substantial reduction of CBP-like immunoreactivity (CBP-LI) in the dorsal columns and a reduction of CM- and PV-LI in the ventromedial dorsal horn. In the motor system, only CM labeled large motoneurons in lamina IX and CB-D28K lightly stained pyramidal tract. The apparent absence of CM-LI in the superficial dorsal horn is contradictory to the presence of a CM-dependent nitric oxide synthase in the region. These data indicate that most CBP-LI in the dorsal column pathway had primary afferent origin, while the superficial dorsal horn exhibited intrinsic CBP immunoreactivity. The differential and selective localizations of CBPs in the spinal cord suggest a role for these proteins in spinal nociceptive processing, visceral regulation and dorsal column sensory pathways.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Médula Espinal/metabolismo , Animales , Calbindina 1 , Calbindina 2 , Calbindinas , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/inmunología , Calmodulina/química , Calmodulina/inmunología , Calmodulina/metabolismo , Humanos , Parvalbúminas/química , Parvalbúminas/inmunología , Parvalbúminas/metabolismo , Ratas , Proteína G de Unión al Calcio S100/química , Proteína G de Unión al Calcio S100/inmunología , Proteína G de Unión al Calcio S100/metabolismo , Médula Espinal/anatomía & histología , Médula Espinal/químicaRESUMEN
Parvalbumin is a calcium-binding protein found in fast-twitch skeletal muscles and selected cells in the brain. In several dystrophic mutants in the mouse, the parvalbumin content of skeletal muscles and brain is reduced and this deficiency appears to correlate with the inability of these mice to handle enhanced calcium uptake associated with the dystrophic process. In this study, two overlapping cDNA clones of 392 and 1268 base pairs were isolated from a mouse cDNA library in lambda gt11, characterized, and used as probes to study the involvement of the parvalbumin gene and its expression in various tissues of dystrophic mice of strain 129 ReJ. Southern blot analyses of restriction fragments of genomic DNA from normal and dystrophic mice indicate the same number and size of parvalbumin-specific gene fragments observed by other researchers, suggesting that the size of the Pva gene is the same in both normal and dystrophic mice of strain 129 ReJ. Northern blot analyses of total RNA from hind-limb muscles using cloned parvalbumin cDNA as probes revealed an abundant 800-nucleotide mRNA with lesser amounts of a 1000-nucleotide mRNA transcript in both normal and dystrophic mice of strain 129 ReJ. The amount of these mRNAs was reduced by 65-77% in dystrophic muscles preparations and was similar to the levels of beta-actin mRNA in these animals. These results suggest that parvalbumin gene expression is not down regulated in dystrophic mice of strain 129 ReJ.
Asunto(s)
ADN Complementario/genética , Expresión Génica , Distrofia Muscular Animal/genética , Parvalbúminas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Sondas de ADN , Enzimas de Restricción del ADN , ADN Complementario/química , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Parvalbúminas/análisis , Parvalbúminas/química , Análisis de Secuencia de ADNRESUMEN
A weak Ca(2+)-binding site (Ka = 0.8 x 10(3) M-1, at pH 7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236-->Ile) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca2+ (> 2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding-folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.
Asunto(s)
Bacillus subtilis/metabolismo , Calcio/metabolismo , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Parvalbúminas/química , Secuencia de Aminoácidos , Bacillus subtilis/citología , Bacillus subtilis/genética , Sitios de Unión , Membrana Celular/metabolismo , Pared Celular/metabolismo , Pared Celular/ultraestructura , Variación Genética , Hexosiltransferasas/biosíntesis , Cinética , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Time-resolved fluorescence spectroscopy permits the direct assessment of proteins motions in the picosecond-nanosecond time-scaled, i.e., in a time-window compatible with observation of relevant motions of the protein matrix. The intrinsic fluorescence emission from tryptophan and tyrosine residues provides a convenient tool to follow these dynamic events in proteins. In the present investigation, the use of time-resolved fluorescence spectroscopy to monitor protein dynamics is illustrated by a study of the effects of temperature and calcium binding on the internal dynamics of the calcium-binding protein, parvalbumin, and by an investigation of the effects of hydration on the measurements of both fluorescence intensity and anisotropy decays provided complementary information regarding the flexibility of aromatic side chains in the proteins investigated, which could be correlated with environmental effects on protein structure.