RESUMEN
Actomyosin precipitation is a critical step in the purification of myosins. In this work, the objective was to precipitate rat kidney actomyosin and isolate myosin by freezing and thawing the soluble fraction. Kidney was homogenized in imidazole buffer, centrifuged at 45000 g for 30 min, and the supernatant was frozen at -20°C for 48 h. The supernatant was thawed at 4°C, centrifuged at 45000 g for 30 min and the precipitate washed twice with imidazole buffer pH 7.0 (with and without Triton X-100, respectively). The resulting precipitate presented a polypeptide profile in SDS/PAGE characteristic of actomyosin and expressed Mg- and K/EDTA-ATPase activity. The actomyosin complex was solubilized with ATP and Mg, and the main polypeptide, p200, was purified in a DEAE-Sepharose column. p200 was marked with anti-myosin II, co-sedimented with F-actin in the absence, but not in the presence, of ATP and was identified by MS/MS with a high Mascot score for myosin IIA. The analysis identified peptides exclusive of myosin IIB, but detected no peptides exclusive of myosin IIC.
Asunto(s)
Congelación , Riñón/metabolismo , Miosina Tipo IIA no Muscular/aislamiento & purificación , Miosina Tipo IIB no Muscular/aislamiento & purificación , Actinas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Animales , Precipitación Química , Cromatografía por Intercambio Iónico , Magnesio/química , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIB no Muscular/química , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We have found that MLC-dependent activation of myosin IIB in migrating cells is required to form an extended rear, which coincides with increased directional migration. Activated myosin IIB localizes prominently at the cell rear and produces large, stable actin filament bundles and adhesions, which locally inhibit protrusion and define the morphology of the tail. Myosin IIA forms de novo filaments away from the myosin IIB-enriched center and back to form regions that support protrusion. The positioning and dynamics of myosin IIA and IIB depend on the self-assembly regions in their coiled-coil C terminus. COS7 and B16 melanoma cells lack myosin IIA and IIB, respectively; and show isoform-specific front-back polarity in migrating cells. These studies demonstrate the role of MLC activation and myosin isoforms in creating a cell rear, the segregation of isoforms during filament assembly and their differential effects on adhesion and protrusion, and a key role for the noncontractile region of the isoforms in determining their localization and function.
Asunto(s)
Actomiosina/fisiología , Movimiento Celular/fisiología , Miosina Tipo IIB no Muscular/fisiología , Animales , Células CHO , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Cricetinae , Cricetulus , Haplorrinos , Humanos , Melanoma , Cadenas Ligeras de Miosina/fisiología , Miosina Tipo IIA no Muscular/fisiología , Miosina Tipo IIB no Muscular/aislamiento & purificación , FosforilaciónRESUMEN
We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDI-TOF MS) and sequenced by liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified.