Caracterização cinética da fosfatase alcalina durante o processo de ossificação em Lithobates catesbeianus
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Data
2019-02-27
Autores
Colosio, Rafael Rodrigues
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Universidade Estadual Paulista (Unesp)
Resumo
Os processos de formação, crescimento, remodelação e, quando necessário, reparo do tecido ósseo ocorrem pela ação coordenada e regulada dos eventos químicos e fisiológicos que participam das ossificações intramembranosa e endocondral, sendo assim, o desequilíbrio das biomoléculas envolvidas no processo, pode levar ao desenvolvimento de patologias ósseas. Durante a metamorfose dos anuros, ocorrem acentuadas e perceptíveis alterações morfológicas que possibilitam a transição do animal do ambiente aquático para o terrestre, sendo a remodelação do esqueleto uma das transformações mais notáveis. Dentre as formas de se estudar o mecanismo de calcificação biológica, as enzimas fosfatase alcalina e fosfatase ácida tartarato-resistente são utilizadas por serem consideradas marcadores bioquímicos da ação dos osteoblastos e dos osteoclastos, respectivamente. Neste sentido, foram realizados estudos em girinos e rãs de Lithobates catesbeianus com o objetivo de se compreender melhor tais processos de ossificação. A média da atividade específica para a hidrólise do pNFF (pH=10,5) pela fosfatase alcalina solubilizada por fosfatidilinositol fosfolipase C-específica (PIPLC) de Bacillus cereus, entre as diferentes regiões ósseas nas diferentes idades do animal, foi de 1142,57 U.mg-1, enquanto que para a hidrólise do PPi (pH=8,0) foi de 1433,82 U.mg-1. Dentre os compostos testados sobre a atividade enzimática, aquele que mais influenciou foi o EDTA, com aproximadamente 67% de inibição para a atividade de pNFFase, e 77% para a de PPase. No caso dos parâmetros cinéticos, a enzima apresentou comportamento “Michaeliano” para a hidrólise do pNFF, bem como para o PPi. O valor de Km ficou em torno de 0,6 mM para a atividade de pNFFase e variou de 0,01 a 0,11 para a atividade de PPase, indicando que a enzima possui maior afinidade por este substrato. O estudo da hidrólise do pNFF e do PPi pela enzima revelou que o pH ótimo de atuação para o pNFF foi de 10,5, enquanto o do PPi, o qual é considerado o verdadeiro substrato da fosfatase alcalina, foi de 8,0, próximo ao fisiológico. Os resultados obtidos mostram que independentemente do tipo de ossificação que ocorre, trata-se da mesma enzima ou isoenzimas atuando nas diferentes regiões ósseas e nas diferentes fases de vida dos anuros. Além disso, o início da formação do osso ocorre na diáfise até o final da metamorfose e segue em direção às epífises quando o animal atinge a fase adulta. Tal fato pode ser explicado porque na fase aquática os ossos não necessitam ser tão rígidos quanto na terra, onde o efeito do impacto direto sobre este tecido provoca uma adaptação mecânica devido ao estímulo externo, logo, o animal precisará de membros reforçados para poder se locomover através de saltos. A semelhança dos resultados com trabalhos realizados com outros vertebrados demonstra que os anfíbios podem ser considerados ótimos modelos animais para o estudo da calcificação biológica.
The processes of formation, growth, remodeling and, when necessary, repair of the bone tissue occur through the coordinated and regulated action of the chemical and physiological events that participate in the intramembranous and endochondral ossifications, thus, the imbalance of the biomolecules involved in the process,maylead to the development of bone pathologies. During anuran’s metamorphosis, there are marked and perceptible morphological changes that allow thetransition of the animal from the aquatic to the terrestrial environment, and the skeleton remodeling is one of the most remarkable transformations. Among the ways to study the mechanism of biological calcification, the enzymes alkaline phosphatase and tartrate-resistant acid phosphatase are used as biochemical markers of the action of osteoblasts and osteoclasts, respectively. In this sense, studies were carried out on tadpoles and frogs of Lithobates catesbeianusin order to better understand such ossification processes. The mean of the specific activity for the hydrolysis of pNPP(pH=10.5) by the alkaline phosphatase solubilised by Bacilluscereusphosphatidylinositol-specific phospholipase C (PIPLC)between the different bone regions at different ages of the animal was 1142.57 U.mg-1, while for the hydrolysis of PPi (pH=8.0) was 1433.82 U.mg-1. Among the compounds tested on the enzymatic activity, the one that most influenced was EDTA, with approximately 67% inhibition for pNPPase activity, and77% for PPase. In the case of kinetic parameters, the enzyme presented "Michaelian" behavior for the hydrolysis of pNPP, as well as for PPi. The Kmvalue was around 0.6 mM for pNPPase activity and ranged from 0.01 to 0.11 for PPase activity, indicating that the enzyme has higher affinity for this substrate. The studyof the hydrolysis of pNPPand PPi by the enzyme revealed that the optimum pH for pNPPwas 10.5, while that forPPi, which is considered the naturalsubstrate of alkaline phosphatase, was 8.0, beingcloser to the physiological. The obtained results show that, regardless of the ossification type that occurs, it is the same enzyme or isoenzymes actingin different bone regions and in the different life phasesof the anurans. In addition,the beginning ofbone formation occursin the diaphysis until the metamorphosis end and,follows towards the epiphysiswhen the animal reaches the adult phase. This fact can be explainedbecausein the aquatic phase the bones do not need to be so rigidas in land, where the effect of the direct impact on this tissue causes a mechanical adaptation due to the external stimulus, so the animal will need reinforced limbs to be able to move through jumps.The similarity of the results to studies with other vertebrates shows that amphibians can be consideredgood animal models for the study of biological calcification.
The processes of formation, growth, remodeling and, when necessary, repair of the bone tissue occur through the coordinated and regulated action of the chemical and physiological events that participate in the intramembranous and endochondral ossifications, thus, the imbalance of the biomolecules involved in the process,maylead to the development of bone pathologies. During anuran’s metamorphosis, there are marked and perceptible morphological changes that allow thetransition of the animal from the aquatic to the terrestrial environment, and the skeleton remodeling is one of the most remarkable transformations. Among the ways to study the mechanism of biological calcification, the enzymes alkaline phosphatase and tartrate-resistant acid phosphatase are used as biochemical markers of the action of osteoblasts and osteoclasts, respectively. In this sense, studies were carried out on tadpoles and frogs of Lithobates catesbeianusin order to better understand such ossification processes. The mean of the specific activity for the hydrolysis of pNPP(pH=10.5) by the alkaline phosphatase solubilised by Bacilluscereusphosphatidylinositol-specific phospholipase C (PIPLC)between the different bone regions at different ages of the animal was 1142.57 U.mg-1, while for the hydrolysis of PPi (pH=8.0) was 1433.82 U.mg-1. Among the compounds tested on the enzymatic activity, the one that most influenced was EDTA, with approximately 67% inhibition for pNPPase activity, and77% for PPase. In the case of kinetic parameters, the enzyme presented "Michaelian" behavior for the hydrolysis of pNPP, as well as for PPi. The Kmvalue was around 0.6 mM for pNPPase activity and ranged from 0.01 to 0.11 for PPase activity, indicating that the enzyme has higher affinity for this substrate. The studyof the hydrolysis of pNPPand PPi by the enzyme revealed that the optimum pH for pNPPwas 10.5, while that forPPi, which is considered the naturalsubstrate of alkaline phosphatase, was 8.0, beingcloser to the physiological. The obtained results show that, regardless of the ossification type that occurs, it is the same enzyme or isoenzymes actingin different bone regions and in the different life phasesof the anurans. In addition,the beginning ofbone formation occursin the diaphysis until the metamorphosis end and,follows towards the epiphysiswhen the animal reaches the adult phase. This fact can be explainedbecausein the aquatic phase the bones do not need to be so rigidas in land, where the effect of the direct impact on this tissue causes a mechanical adaptation due to the external stimulus, so the animal will need reinforced limbs to be able to move through jumps.The similarity of the results to studies with other vertebrates shows that amphibians can be consideredgood animal models for the study of biological calcification.
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Palavras-chave
Tecido ósseo, Calcificação biológica, Fosfomonohidrolases, Rã-touro, Metamorfose