Identificação e caracterização de isoformas de fosfomonohidrolases presentes na cauda de girinos de rã-touro (Lithobates catesbeianus) durante o desenvolvimento larval
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Data
2017-03-15
Autores
Gonçalves, Adriano Marques [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A metamorfose dos anfíbios é um processo altamente regulado que envolve a participação de hormônios e biomoléculas na morte celular e absorção da cauda, a qual libera nutrientes para as transformações morfofisiológicas necessárias para a ocupação do meio terrestre. Dentre os nutrientes liberados, o fosfato é essencial para a síntese de ATP, membranas biológicas, ácidos nucleicos e metabólitos fosforilados, bem como para a regulação de processos degradativos e da atividade de enzimas. Neste sentido, a identificação e caracterização cinética das fosfatases que participam da absorção da cauda de girinos de Lithobates catesbeianus, durante a passagem da fase larval aquática para a adulta terrestre, poderão contribuir para a compreensão dos eventos envolvidos na morte celular e na liberação de nutrientes. O estudo revelou aumento da atividade das fosfatases ácida e alcalina, de aproximadamente 30 e 17 vezes, respectivamente, no período da metamorfose. A centrifugação diferencial do extrato bruto da cauda e os estudos cinéticos, permitiram a identificação de três fosfatases distintas, sendo uma fosfatase ácida solúvel, uma fosfatase ácida ligada à membrana e uma fosfatase alcalina ligada à membrana por meio da âncora de fosfatidilinositol. A fosfatase ácida ligada à membrana revelou Vm=104,5 U.mg-1; Km=0,29 mM e nH=0,9; a solúvel, Vm= 25,9 U.mg-1; Km=0,33 mM e nH =0,8, enquanto a fosfatase alcalina ligada à membrana revelou Vm=10,5 U.mg-1; Km=0,2 mM e nH=1,0, para a hidrólise do pNPP. Os resultados da espectrometria de massas revelaram a presença de três proteína fosfatases, sendo duas serina/treonina, uma PP2A, uma PP1 e uma tirosina, PTP-LMW. Estudos utilizando substratos específicos para PP2A e PTP-LMW e inibidor para PP1 permitiram confirmar suas presenças na cauda dos girinos de L. catesbeianus. Além disso, a separação em diferentes frações proteicas possibilitou atribuir atividade de PP2A mitocondrial à fosfatase alcalina de membrana, de PTP-LMW à fosfatase ácida solúvel e de PP1 à fosfatase ácida de membrana. Os resultados obtidos levaram à proposta de um modelo de atuação das referidas enzimas no processo de absorção da cauda de anuros durante a metamorfose. Tal modelo propõe a possível função tanto da PP1, quanto da PP2A na sinalização dos eventos que levam à morte celular, bem como o da PTP-LMW na regulação da proliferação celular.
Amphibian metamorphosis is a tightly regulated transformation that involves the participation of hormones and other biomolecules in cell death and tail absorption, to release nutrients for the morphophysiological changes necessary for the occupation of the terrestrial environment. Among these nutrients, the phosphate is essential for the synthesis of ATP, biological membranes, nucleic acids and phosphorylated metabolites; regulation of degradative processes and of enzymes activity. In this sense, the identification and characterization of the phosphatases that participate in the absorption of the tail of Lithobates catesbeianus during the transition from the aquatic larval phase to the terrestrial adult, may contribute to the understanding of the events involved in cell death and nutrient release.The study showed that there was an increase in the activity of the acid and alkaline phosphatases of approximately 30 and 17 fold, respectively, in the metamorphosis period. The differential centrifugation of the tail crude extract, as well as the kinetic tests, allowed the identification of three distinct phosphatases, a soluble acid phosphatase, a membrane bound acid phosphatase and an alkaline phosphatase bound to the membrane by the anchor of phosphatidylinositol. The kinetic characterization of membrane bound acid phosphatase revealed Vm=104.5 U.mg-1; K0.5=0.29 mM and nH=0.9; the soluble, Vm= 25.9 U.mg-1; K0.5=0.33 mM and nH =0.8, while the membrane bound alkaline phosphatase revealed Vm=10.5 U.mg-1; K0.5=0.2 mM and nH=1.0, for pNPP hydrolysis. The mass spectrometry results showed the presence of three protein phosphatases, two serine/threonine, being one PP2A, one PP1 and one tyrosine, PTP-LMW. Studies using specific substrates for PP2A and PTP-LMW and inhibitor for PP1 confirmed their presence in the tail of the tadpoles of L. catesbeianus. In addition, the separation in different protein fractions, allowed to attribute PP2A mitochondrial activity to the membrane alkaline phosphatase, PTP-LMW to the soluble acid phosphatase and PP1 to the membrane acid phosphatase. A model of the role of these enzymes in the process of anurans tail absorption, during metamorphosis, is proposed. Such model provides information on the role of both PP1 and PP2A in the signaling events leading to cell death, as well as that of PTP-LMW in the regulation of cell proliferation.
Amphibian metamorphosis is a tightly regulated transformation that involves the participation of hormones and other biomolecules in cell death and tail absorption, to release nutrients for the morphophysiological changes necessary for the occupation of the terrestrial environment. Among these nutrients, the phosphate is essential for the synthesis of ATP, biological membranes, nucleic acids and phosphorylated metabolites; regulation of degradative processes and of enzymes activity. In this sense, the identification and characterization of the phosphatases that participate in the absorption of the tail of Lithobates catesbeianus during the transition from the aquatic larval phase to the terrestrial adult, may contribute to the understanding of the events involved in cell death and nutrient release.The study showed that there was an increase in the activity of the acid and alkaline phosphatases of approximately 30 and 17 fold, respectively, in the metamorphosis period. The differential centrifugation of the tail crude extract, as well as the kinetic tests, allowed the identification of three distinct phosphatases, a soluble acid phosphatase, a membrane bound acid phosphatase and an alkaline phosphatase bound to the membrane by the anchor of phosphatidylinositol. The kinetic characterization of membrane bound acid phosphatase revealed Vm=104.5 U.mg-1; K0.5=0.29 mM and nH=0.9; the soluble, Vm= 25.9 U.mg-1; K0.5=0.33 mM and nH =0.8, while the membrane bound alkaline phosphatase revealed Vm=10.5 U.mg-1; K0.5=0.2 mM and nH=1.0, for pNPP hydrolysis. The mass spectrometry results showed the presence of three protein phosphatases, two serine/threonine, being one PP2A, one PP1 and one tyrosine, PTP-LMW. Studies using specific substrates for PP2A and PTP-LMW and inhibitor for PP1 confirmed their presence in the tail of the tadpoles of L. catesbeianus. In addition, the separation in different protein fractions, allowed to attribute PP2A mitochondrial activity to the membrane alkaline phosphatase, PTP-LMW to the soluble acid phosphatase and PP1 to the membrane acid phosphatase. A model of the role of these enzymes in the process of anurans tail absorption, during metamorphosis, is proposed. Such model provides information on the role of both PP1 and PP2A in the signaling events leading to cell death, as well as that of PTP-LMW in the regulation of cell proliferation.
Descrição
Palavras-chave
Morte celular, Metamorfose, Fosfatases, Mitocôndria, Anuro, Cell death, Metamorphosis, Mitochondria, Phosphatases, Anuran