RESUMEN
The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain.
Asunto(s)
Exoesqueleto/metabolismo , Bivalvos/genética , Quitina Sintasa/genética , Quitina/biosíntesis , Nácar/metabolismo , Secuencia de Aminoácidos , Animales , Bivalvos/clasificación , Bivalvos/enzimología , Pollos , Quitina Sintasa/química , Quitina Sintasa/metabolismo , Agua Dulce , Expresión Génica , Glucosiltransferasas/química , Glucosiltransferasas/genética , Punto Isoeléctrico , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Sistemas de Lectura Abierta , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg(2+)ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS). This reaction is sensitive to conformational changes in the helix-loop-helix and the labeling efficiency was reduced when S1 was bound to actin, DNP or nucleotide analogs. The nucleotide analogs had a range of effects (PPi>ADP·AlF(4)(-)>ADP) irrespective of the open-closed state of switch 2. The greatest reduction in labeling was in the presence of actin or DNP. When we measured the effect of each ligand on the fluorescence of FHS previously attached to S1, only DNP quenched the emission. Together, the results suggest that the helix-loop-helix region is flexible, it is part of the communication pathway between the ATP- and actin-binding sites of myosin and it is proximal to the region of myosin where DNP binds.
Asunto(s)
2,4-Dinitrofenol/farmacología , Colorantes/farmacología , Lisina/metabolismo , Subfragmentos de Miosina/metabolismo , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Fluorescencia , Colorantes Fluorescentes , Secuencias Hélice-Asa-Hélice , Subfragmentos de Miosina/química , Unión Proteica , Conformación Proteica/efectos de los fármacos , ConejosRESUMEN
We have demonstrated previously that urea inhibits the activity and alters the tertiary structure of skeletal muscle myosin in a biphasic manner. This was attributed to differential effects on its globular and filamentous portion. The inhibition of catalytic activity was counteracted by methylamines. With the aim of comprehending the effects of urea on the catalytic (globular) portion of myosin, this study examines the effects of urea and the countereffects of betaine on the catalytic activity and structure of myosin subfragment-1. It is shown that urea inactivates subfragment-1 in parallel with its ability to induce exposure of the enzyme hydrophobic domains, as assessed using intrinsic and extrinsic fluorescence. Both effects are counteracted by betaine, which alone does not significantly affect subfragment-1. Urea also enhances the accessibility of thiol groups, promotes aggregation and decreases the alpha-helix content of S1, effects that are also counteracted by betaine. We conclude that urea-induced inactivation of the enzyme is caused by partial unfolding of the myosin catalytic domain.