Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process.

R R Colosio,A M Gonçalves,C C Santana,G C Pavarina,L F J Santos,J M Pizauro

doi:10.1590/1519-6984.251970

Abstract

In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Highlights

The results show that alkaline phosphatase was purified, since there was a decrease in the protein concentration of the sample and an increase in the recovery factor (RF) at each step, as well as an increase in enzymatic activity in the fraction of interest (SPIPLC)
Because it was not possible to dose the proteins nor to detect the activities of alkaline phosphatase in the SPIPLC fraction of the frog diaphysis, only the other three samples were used in the post protein purification assays
The results show statistical differences between enzyme activities considering the bone regions of tadpole and frog, indicating that the bone region with significantly higher values for both enzymes is in the frog epiphysis, followed by tadpole diaphysis, and the lowest values were observed in the tadpole epiphysis

Summary

Introduction

The process of amphibian metamorphosis, characterized by behavioral, morphological, physiological and biochemical changes, is a classic model used to study the transition of vertebrates from an aquatic to a terrestrial environment (Bo et al, 2018; Gao et al, 2018; Kroth et al, 2018; Rigon et al, 2014).Among the alterations, the morphological and the physiological ones are more perceptible, characterized by the regression of structures necessary only to tadpoles, by the transformation of some larval structures into structures necessary for adults, and by the development of structures only essential to adult animals (Bo et al, 2018; Gao et al, 2018).Modifications of the skeletal system, the development of the forelimbs and hindlimbs, are critically important to anurans (Gao et al, 2018; Trueb and Hanken, 1992) to move in the terrestrial environment by jumping (Fabrezi et al, 2017; Pough et al, 2008).The skeleton consists mainly of bone tissue, a specialized type of connective tissue, which is composed of bone matrix (mineralized extracellular material), and cells called osteoblasts, osteocytes and osteoclasts (Junqueira and Carneiro, 2013; Pizauro Junior et al, 2017). The development of long bones occurs from the combination of two osteogenesis mechanisms, in which periosteal intramembranous ossification is accompanied by endochondral ossification (Çiçek et al, 2011; Felisbino and Carvalho, 1999, 2001; Gómez et al, 2017; Junqueira and Carneiro, 2013; Pizauro Junior et al, 2017; Song et al, 2010). This process occurs mainly by periosteal ossification, which advances from the center of the long bones (diaphysis) towards the bone end (epiphysis) faster than the endochondral ossification (Çiçek et al, 2011; Felisbino and Carvalho, 1999, 2001; Fröbisch, 2008; Gómez et al, 2017)

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