RESUMEN
A wide variety of post-translational modifications such as oxidation, phosphorylation, glycosylation, methylation, and acetylation play critical roles in cellular functions. Detection of post-translational modifications in proteins is important to understand their crucial roles in cellular functions. Identifying each modification requires special attention in mass spectral acquisition and analysis. Here, we report a mass spectral method for the detection of multiple phosphorylations in peptides by analyzing their products after fragmentation. Synthetic peptides were used to identify these modifications by matrix-assisted laser desorption/ionization (MALDI) TOF/TOF. Peptides with serine, threonine, and tyrosine were used with mono- to tetra-phosphorylation sites in different combinations to get insights into their fragmentation and identify the location of these sites. The y-ion series were observed without the loss of phosphate groups and were thus very useful in determining the localization and sequence of the phosphate residues. Acetylation of the peptides was found to be useful in detecting the b1-ion and helped in identifying the N-terminus. When a mixture of the phosphorylated peptides (from mouse protein sequences) were analyzed by LC-MS/MS on a Velos Orbitrap Mass Spectrometer and the data subjected to analysis by Sequest using the mouse database, the peptides were identified along with the parent proteins. A comparison of MALDI TOF/TOF spectra with ESI MS/MS helped in eliminating falsely discovered peptides using the database search.
Asunto(s)
Péptidos/química , Fosfatos/química , Espectrometría de Masas en Tándem/métodos , Acetilación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Bases de Datos de Proteínas , Glicosilación , Metilación , Ratones , Oxidación-Reducción , Fosforilación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The role of dihydrolipoamide dehydrogenase (DLD), the E3 subunit of the pyruvate dehydrogenase complex (PDHc) in hamster sperm capacitation and acrosome reaction has been implicated previously. In this study, attempt has been made to understand DLD/PDHc involvement from the perspective of pyruvate/lactate metabolism. Inhibition of DLD was achieved by the use of a specific inhibitor, 5-methoxyindole-2-carboxylic acid. It was seen that 5-methoxyindole-2-carboxylic acid-treated spermatozoa with inhibited DLD (and PDHc) activity had lactate accumulation, which caused an initial lowering of the intracellular pH and calcium and an eventual block in capacitation and acrosome reaction. Collectively, the data reveal a significant contribution of the metabolic enzymes DLD and PDHc to lactate regulation in hamster spermatozoa during capacitation and acrosome reaction. Additionally, the importance of lactate regulation in the maintenance of sperm intracellular pH and calcium, two important physiologic factors essential for sperm capacitation and acrosome reaction, has also been established.
Asunto(s)
Reacción Acrosómica/fisiología , Calcio/metabolismo , Dihidrolipoamida Deshidrogenasa/fisiología , Ácido Láctico/metabolismo , Capacitación Espermática/fisiología , Espermatozoides/enzimología , Animales , Cricetinae , Dihidrolipoamida Deshidrogenasa/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Indoles/farmacología , Masculino , Mesocricetus , Espermatozoides/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate whether the eNOS gene influences the risk of developing endometriosis in south Indian women. STUDY DESIGN: The single nucleotide polymorphism, Glu298Asp, in exon7 of the eNOS gene was tested for association in a case-control study of 232 affected women and 210 women with no evidence of disease. All the women were infertile, ascertained from the same infertility clinic. The genotype frequencies of the polymorphism were compared, using polymerase chain reaction and sequencing analysis. The localization and expression of eNOS in the eutopic endometrium of five cases and four controls was also analyzed using immunohistochemistry and western blotting. RESULTS: No statistically significant differences were observed in the genotype distributions and allele frequencies (p=0.3) between the cases and controls according to codominant, dominant and recessive genotype models. The localization and expression of this protein were similar in the endometrium of cases and controls. CONCLUSION: In the present study we could neither observe a difference in the eNOS expression nor establish an association between the eNOS Glu298Asp exon 7 polymorphism in south Indian women with endometriosis.
Asunto(s)
Endometriosis/genética , Predisposición Genética a la Enfermedad/genética , Óxido Nítrico Sintasa de Tipo III/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Estudios de Casos y Controles , Estudios de Cohortes , Endometriosis/metabolismo , Endometrio/metabolismo , Femenino , Humanos , India , Óxido Nítrico Sintasa de Tipo III/metabolismo , Adulto JovenRESUMEN
Mammalian testicular spermatozoa are immotile and incompetent for fertilization. They acquire motility during epididymal maturation and fertilizing ability during a second phase of maturation in the female reproductive tract, termed as capacitation. Capacitation was discovered independently by Austin and Cang in early 1950s and was defined as the obligate period of residency of spermatozoa in the female reproductive tract, which confers on the spermatozoa the ability to fertilize an oocyte. Over the years, the definition of capacitation has changed and it has been recognized as a complex phenomenon, which is correlated with changes associated with the spermatozoa in the female tract. These alterations in metabolism, intracellular ion concentration, membrane fluidity, intracellular pH, cAMP concentration and concentration of reactive oxygen species, ultimately make the spermatozoa fertilization-competent. The molecular basis of capacitation is poorly understood despite the fact that it is an important event preceding fertilization. This review presents our current understanding of the signaling events involved in the process of capacitation.
Asunto(s)
Reacción Acrosómica/fisiología , Transducción de Señal/fisiología , Capacitación Espermática/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Acrosoma/fisiología , Adenilil Ciclasas/metabolismo , Animales , AMP Cíclico/metabolismo , Femenino , Humanos , Masculino , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Maduración del Esperma , Motilidad EspermáticaRESUMEN
In an attempt to understand the role of nitric oxide(NO) in sperm capacitation, in the present study, hamster spermatozoa were used to evaluate the effects of NO on motility, viability, hyperactivation, capacitation and protein tyrosine and serine phosphorylation using specific inhibitors of nitric oxide synthase (NOS); namely L-NAME (N-nito-L-aginine methyl ester) and 7-Ni (7-nitroindazole). The results indicated that L-NAME inhibits sperm motility, hyperactivation and acrosome reaction where as 7-Ni inhibits only hyperactivation and acrosome reaction thus implying that NOS inhibitors exhibit subtle differences with respect to their effects on sperm functions. This study also provides evidence that NOS inhibitors inhibit sperm capacitation by their ability to modulate protein tyrosine phosphorylation. However, the inhibitors had no effect on the protein serine phosphorylation of hamster spermatozoa during capacitation. Thus, these results indicate that NO is required
Asunto(s)
Indazoles/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/fisiología , Capacitación Espermática/efectos de los fármacos , Espermatozoides/fisiología , Reacción Acrosómica/efectos de los fármacos , Animales , Cricetinae , Inhibidores Enzimáticos/farmacología , Masculino , Mesocricetus , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fosforilación , Proteínas/metabolismo , Motilidad Espermática , Interacciones Espermatozoide-Óvulo , Espermatozoides/efectos de los fármacosRESUMEN
The effects of guanosine tetraphosphate (ppGpp) on inhibition of single-round in vitro transcription and on the kinetics of open complex formation were investigated at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1. The two promoters differ in their saturation characteristics and sensitivities to ppGpp. With a 10:1 molar ratio of RNA polymerase (RNAP) to DNA, saturation of transcription activity and weak inhibition (approximately 30%) are observed at rplJ, in contrast to the weak activity and strong inhibition (approximately 80%) at rpsA P1. In the absence of ppGpp, the two promoters show a threefold difference in the overall rate constants of association (ka) (6.5 x 10(7) M-1 s-1 at rplJ and 2.0 x 10(7) M-1 s-1 at rpsA P1), while the dissociation rate constants (kd) are similar (approximately 4.8 x 10(-5) s-1). The addition of ppGpp causes a twofold reduction in k2 (isomerisation constant) rplJ and a threefold decrease in KB (equilibrium constant of RNAP binding) at rpsA P1. There is a significant twofold increase in kd at rplJ, compared with smaller changes at rpsA P1 and at the non-stringent lacUV5 promoter. These results indicate that ppGpp affects the formation and stability of the open complex at the rplJ promoter, in contrast to the inhibition of RNAP binding to the rpsA P1 promoter.