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

Atividade enzimática de marcadores ósseos no crescimento de Lithobates catesbeianus (Shaw, 1802) durante o processo de ossificação

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 K_m 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.

Keywords:
anura; biological calcification; metamorphosis; phosphatases; kinect characterization

Resumo

Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg^-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg^-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de K_m foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.

Palavras-chave:
anura; calcificação biológica; metamorfose; fosfatases; caracterização cinética

1. 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 easily 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, more specifically 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). Bone formation, as well as fracture repair, occurs through two processes called intramembranous ossification and endochondral ossification (Junqueira and Carneiro, 2013; Song et al., 2010).

In vertebrates in general, 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). In amphibians, 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).

Biological calcification occurs through the formation of hydroxyapatite crystals, requiring the release and transport of inorganic phosphate (Pi) into extracellular matrix vesicles (EMV), which originate from osteoblast membrane surface, being a process performed by the coordinated action of enzymes (Millán, 2013; Pizauro Junior et al., 2017).

Inside the EMV, initial Pi production is performed by PHOSPHO1, which catalyzes the hydrolysis of phosphocholine and phosphoethanolamine by releasing Pi. Such substrates are formed from the hydrolysis of phospholipids: phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, located on the inner side of the vesicle membrane, by the action of phospholipases and sphingomyelinase 2. On the outside of the EMV, PC-1 is responsible for the production of the alkaline phosphatase substrate, PPi, derived from ATP hydrolysis, that is hydrolyzed by alkaline phosphatase and a Pi is released. Finally, calcium is translocated into the vesicle by annexins, which act as calcium channels, while Pi is translocated into the EMV by carriers. Thus, the increase of the calcium versus phosphate ionic product within the vesicles leads to calcium phosphate precipitation, leading to the formation of crystals that perforate the vesicle membrane and spill, exposing preformed hydroxyapatite to the extracellular fluid, allowing the propagation and deposition of hydroxyapatite on collagen fibers present in the extracellular matrix (Millán, 2013; Pizauro Junior et al., 2017).

Among the enzymes involved in the biological calcification process, alkaline phosphatase has been used as a specific biochemical marker for osteoblasts, the cells responsible for bone formation, while tartrate-resistant acid phosphatase has been used as a marker for osteoclasts, the cells responsible for resorption the bone tissue (Gijsbers et al., 2001; Johnson et al., 2000). Therefore, the study of these enzymes may provide important information about the metabolic activity of this tissue.

During the anurans metamorphosis period, the formation and then the growth of the limbs of the animal occur, through both of the ossification processes (Felisbino and Carvalho, 1999, 2001; Gómez et al., 2017). In the case of birds and mammals, when the animal is born, the limbs are already formed, thus only bone growth is observed from then on (Pizauro Junior et al., 2017). Because of this, the tadpoles and frogs of the species Lithobates catesbeianus were chosen as a model to study periosteal and endochondral ossification, processes required not only during bone tissue development and growth, but also during fracture repair (Song et al., 2010). In addition, the study, through the kinetic characterization of alkaline phosphatase released by PIPLC and the activity of acid phosphatase and tartrate-resistant acid phosphatase, and understanding the role of these enzymes may help prevent and/or cure certain bone pathologies (Junqueira and Carneiro, 2013; Pizauro Junior et al., 2017), in anurans and other vertebrates.

The present work characterized the kinetic properties of alkaline phosphatase and studied the activities of tartrate-resistant acid phosphatase and acid phosphatase. These enzymes are associated with mineralization, resorption and bone tissue formation processes. In this sense, the results may contribute to better understand anuran bone formation, as well as to demonstrate the potential of amphibians as excellent animal models for the study of the biological mineralization process.

2. Material and Methods

The animal procedures as well as the manipulation and sacrifice protocols used in this study were approved by the Animal Use Ethics Commission (Comissão de Ética no Uso de Animais – CEUA) of the Faculty of Agriculture and Veterinary Sciences (Faculdade de Ciências Agrárias e Veterinárias – FCAV), protocol No.10315/15, and are therefore in accordance with the Ethical Principles on Animal Experimentation (Princípios Éticos na Experimentação Animal), adopted by the Brazilian College of Animal Experimentation (Colégio Brasileiro de Experimentação Animal).

The experiments were carried out at the Frog Farming Sector of the Aquaculture Center of UNESP (CAUNESP), and at the Laboratory of Applied Enzymology and Immunochemistry (Laboratório de Enzimologia e Imunoquímica Aplicadas – LEIA) of the Technology Department of UNESP, Campus of Jaboticabal in São Paulo, Brazil.

2.1. Experimental animals

Bullfrog (Lithobates catesbeianus) tadpoles were kept in tanks with 2,000 L of capacity and a density of one tadpole per 2 L of water, with an average temperature of 27 °C. For food, a commercial ration was provided three times a day until apparent satiation. Tadpoles (n≈500) were collected between stages 42 and 46 (metamorphic climax), according to Gosner (1960) table, during which the largest limb development occurs.

The frogs were housed in bays of approximately 12 m², containing shelter sites, central water chutes and linearly arranged vibratory feeders. Continuous flow of water was provided from an artesian well. A commercial ration was provided. According to the needs of the experiment frogs were collected (n≈50), in growth phase, approximately six months after metamorphosis (from 9 months to 1 year old).

2.2. Slaughtering animals and obtaining bones for the production of enzyme extracts

To obtain the enzyme extracts, the animals were desensitized in water with ice at ≈4 °C, the tadpoles were decapitated (Czesnik et al., 2006), by separating the head from the body, and the frogs were submitted to a concussion of the spinal marrow. The hind limbs were then collected, immediately frozen in liquid nitrogen, and stored separately at -70 °C.

After thawing, the soft and adherent tissues were removed from the femurs. Then, bone marrow was removed, and the femurs were divided into epiphysis and diaphysis, ground separately in a mill-type appliance (TECNAL brand, model TE 631), and then homogenized in a TURRAX type apparatus (OMNI brand, model GLH-2511), in TRIS.HCl buffer 5 mM, pH=7.5 containing 2 mM MgCl₂ and 1 µM ZnCl₂, in the proportion of 1 g of tissue to 5 mL of buffer. The homogenate was centrifuged at 10,000 g for 10 minutes at 4 °C (see Supplementary Figure 1). Thereafter, a predetermined amount of supernatant (SEB) was aliquoted, frozen in liquid nitrogen and stored at -70 °C for use in subsequent assays. The remaining SEB was used for differential centrifugation.

2.3. Differential centrifugation to obtain cell membranes

The supernatant (CES) was again centrifuged at 100,000 g for 2 h at 4 °C. Then the supernatant (S1) was aliquoted, and the precipitate (P1) was solubilized in a POTTER type homogenizer with 5 mM TRIS.HCl buffer, pH=7.5 containing 2 mM MgCl₂ and 1 µM ZnCl₂ (see Supplementary Figure 1). A predetermined amount of P1 was aliquoted and the remaining was used in the next purification steps.

2.4. Removal of cell membrane-bound proteins via phosphatidylinositol anchor

Sample P1 was incubated with phospholipase C (1 U PIPLC/mL sample) in 5 mM TRIS.HCl buffer, pH=7.5, containing 2 mM MgCl₂ and 1 µM ZnCl₂, at 37 °C for 1 h, while kept under constant agitation (Pizauro Junior et al., 1995). After the incubation period, the mixture was centrifuged at 100,000 g for two hours at 4 °C. The obtained supernatant was frozen and stored. The precipitate was solubilized in a POTTER-type homogenizer using the same initial volume of 5 mM TRIS.HCl buffer, pH=7.5, containing 2 mM MgCl₂ and 1 µM ZnCl₂ (Pizauro Junior et al., 1988) (see Supplementary Figure 1). The precipitate and the supernatant were used to evaluate the type of enzyme interaction with the membrane.

2.5. Determination of enzymatic activities

The p-nitrophenylphosphatase (pNPPase) activity of phosphatases was determined discontinuously at 37 °C through the formation of the p-nitrophenolate ion (ε=17600M^-1cm^-1, pH=13), in 100mM AMPOL buffer, pH=10.5 (alkaline phosphatase) containing 2 mM MgCl₂, or 100 mM Acetate buffer, pH=5.0 (acid phosphatase and tartrate resistant acid phosphatase), both containing 1 mM pNPP in a volume of 1 mL. In the case of tartrate-resistant acid phosphatase, sodium tartrate (100mM) was added to the acetate buffer. The reaction was always initiated by the addition of the enzyme to the reaction medium and stopped by the addition of 1 mL of 1 M NaOH at previously established times. Then, the measurement was performed on a Hitachi® U-2000 spectrophotometer at 410 nm.

The pyrophosphatase activity of alkaline phosphatase (PPase) was determined discontinuously at 37 °C by measuring the phosphate released according to the method described by Heinonen and Lahti (1981). The reaction was initiated by the addition of the enzyme to the reaction medium containing 100 mM TRIS.HCl buffer, pH=8.0, in a final volume of 1 mL. At pre-established times the reaction was stopped by the addition of 0.5 mL of cold TCA 30% (w/v) solution and 0.5 mL aliquots were used for measuring the inorganic phosphate released. Then, the measurement was performed on a Hitachi® U-2000 spectrophotometer at 355 nm.

The determinations were performed in triplicates with initial velocities remaining constant for at least 90 minutes with less than 5% of the substrate being hydrolyzed. Controls without enzyme addition were included in each experiment to estimate non-enzymatic substrate hydrolysis.

One unit of enzymatic activity was defined and expressed as the amount of enzyme that releases one nmol of p-nitrophenolate (pNPPase activities) or phosphate (PPase activity) per minute per milligram of protein (U.mg^-1) under the standard test conditions.

2.6. Effect of pH on enzymatic activities in the SPIPLC fraction

The effect of pH on the pNPPase and PPase activities of PIPLC-released alkaline phosphatase was studied using the buffer solutions: TRIS.HCl (pH=6.5-8.5) and AMPOL (pH=8.5-11.0) at a concentration of 100 mM.

2.7. Effect of substrate concentration on enzymatic activities in the SPIPLC fraction

The hydrolysis effect of pNPP and PPi by PIPLC-released alkaline phosphatase was studied using AMPOL (100 mM, pH=10.5, containing 2 mM MgCl₂) and TRIS.HCl (100 mM, pH=8) buffers, respectively, and by varying the concentrations of pNPP (0.01 to 20 mM) and PPi (0.001 to 2 mM).

2.8. Action of compounds on enzymatic activities in the SPIPLC fraction

The study of the effect of different compounds (Levamisole, Theophylline, Zinc, Phosphate, and EDTA) on the pNPPase and PPase activities of the alkaline phosphatase was performed using concentrations ranging from 100 µM to 100 mM, depending on the compound.

2.9. Protein dosage

Protein concentration (mg/mL) was determined by the method proposed by Bradford (1976) using a commercial Bio-Rad® kit following manufacturer's specifications using bovine serum albumin (BSA) as a protein standard.

Since the compounds were evaluated in punctual concentrations, they were compared in percentage of activity with controls.

2.10. Analysis of results

Statistical analyzes were performed using Assistat software version 7.7 beta (Silva and Azevedo, 2016). All results were subjected to analysis of variance. Error normality was evaluated (Kolmogorov-Smirnov) to ensure the assumption of the analysis. Given the significant interaction between the factors, the development was performed. Observing a significant difference between the variables analyzed, the averages of the splits were compared using the Tukey test at a 5% probability. GraphPad Prism (version 5.01) was used to estimate the values of V_max and K_m and Hill coefficient.

3. Results

Table 1 shows protein concentration (mg/mL), recovery factor (RF), and enzymatic activities (ATV) in all fractions during protein purification.

Table 1
Protein concentration, enzymatic activities (U.mg^-1) and recovery factors in protein purification fractions, in the epiphysis and diaphysis of tadpoles and frogs of Lithobates catesbeianus.

Regions/Fractions	Protein (mg/mL)	pNPPase		PPase		Ac.P.		T.R.Ac.P.
		ATV	RF	ATV	RF	ATV	RF	ATV	RF
Tadpole Epiphysis
CES	4.13±0.13	94.42±3.97	1.00	47.00±1.36	1.00	45.65±1.35	1.00	22.90±0.94	1.00
S1	3.84±0.01	42.31±0.25	0.45	20.64±0.68	0.44	19.91±0.45	0.44	7.15±0.18	0.31
P1	0.35±0.02	622.01±24.07	6.59	408.29±13.18	8.69	305.28±11.26	6.69	190.78±3.53	8.33
SPIPLC	0.06±0.00	675.98±19.44	7.16	685.56±15.03	14.59	32.77±0.48	0.72	ND	ND
PPIPLC	0.25±0.00	794.23±11.83	8.41	428.30±33.54	9.11	348.06±7.76	7.63	213.68±4.69	9.33
Tadpole Diaphysis
CES	3.40±0.26	124.90±2.20	1.00	77.68±2.69	1.00	35.85±1.48	1.00	16.57±0.52	1.00
S1	3.14±0.12	52.98±1.74	0.42	39.04±1.78	0.50	20.78±1.30	0.58	6.62±0.38	0.40
P1	0.15±0.01	1,249.12±19.42	10.00	736.45±27.17	9.48	248.92±14.15	6.94	115.99±9.51	7.00
SPIPLC	0.03±0.00	1,028.08±15.43	8.23	1,632.94±23.53	21.02	29.59±0.84	0.83	21.59±1.83	1.30
PPIPLC	0.07±0.00	1,911.16±11.19	15.30	1,532.56±20.13	19.73	222.83±5.56	6.22	116.56±14.02	7.03
Frog Epiphysis
CES	0.68±0.03	221.45±7.29	1.00	219.14±0.77	1.00	38.25±1.42	1.00	20.03±1.33	1.00
S1	0.58±0.01	80.34±2.48	0.36	68.62±0.83	0.31	23.99±0.67	0.63	15.63±0.58	0.78
P1	0.05±0.00	2,174.30±57.59	9.82	1,327.37±6.15	6.06	113.86±4.54	2.98	37.42±12.43	1.87
SPIPLC	0.03±0.00	1,723.65±30.31	7.78	1,982.97±83.96	9.05	13.27±0.88	0.35	ND	ND
PPIPLC	0.04±0.00	2,742.73±19.23	12.39	1,072.35±14.07	4.89	109.94±4.81	2.87	32.87±8.01	1.64
Frog Diaphysis
CES	0.17±0.03	0.819±0.10	1.00	ND	ND	3.644±0.06	1.00	1.146±0.09	1.00
S1	0.15±0.01	0.470±0.03	0.57	ND	ND	3.423±0.09	0.94	1.058±0.05	0.92
P1	ND	ND	ND	ND	ND	ND	ND	ND	ND
SPIPLC	ND	ND	ND	ND	ND	ND	ND	ND	ND
PPIPLC	ND	ND	ND	ND	ND	ND	ND	ND	ND

• pNPPase = PIPLC-released alkaline phosphatase p-nitrophenylphosphatase activity; Ppase = alkaline phosphatase pyrophosphatase activity; Ac.P. = acid phosphatase; T.R.Ac.P. = Tartrate Resistant Acid Phosphatase; ATV = Enzyme Activity; RF = Recovery Factor; ND = Not Detected.

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) (see Table 1).

The present work also aimed to study the kinetic activities of alkaline phosphatase, so after protein purification, the SPIPLC fraction of each sample was used to continue the studies. Thus, 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 (see Table 1), only the other three samples were used in the post protein purification assays.

The analysis of variance shows an association between enzyme activity and bone regions (see Table 2), with rising activities in the following order: Tadpole Epiphysis (680.77 nmol/min/mg), Tadpole Diaphysis (1,330.51 nmol/min/mg) and Frog Epiphysis (1,853.31 nmol/min/mg). The results show statistical differences between enzyme activities considering the bone regions of tadpole and frog (see Table 3), 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. In this sense, according to the statistical analysis, shown in Table 3, both pNPPase and PPase activities differed when all bone regions are compared.

Table 2
ANOVA and results of the comparison of averages of specific activity of pNFFase and PPase of alkaline phosphatase (nmol/min/mg), in the SPIPLC fraction of protein purification, of the tadpole bone regions (epiphysis and diaphysis) and frog (epiphysis).

Factors			Specific activity (nmol/min/mg)
Enzymatic activities	Bone regions
pNPPase			1,142.57
PPase			1,433.82
	Tadpole Epiphysis		680.77
	Tadpole Diaphysis		1,330.51
	Frog Epiphysis		1,853.31
Analysis of variance: p values
Enzymes		<0.001**
Bone regions		<0.001**
Enzymes X Bone regions		<0.001**
VC%		3.07%

• ^**
  significant at the 1% probability level; VC% = variation coefficient.

Table 3
Developments of enzyme activities and bone regions. Averages of pNPPase and PPase specific activity of alkaline phosphatase (nmol/min/mg) in protein purification of the SPIPLC fraction of tadpole and frog bone tissue regions.

Enzymatic activities	Bone regions
	Tadpole Epiphysis	Tadpole Diaphysis	Frog Epiphysis
pNPPase	675.98±19.44C	1,028.08±15.43bB	1,723.65±30.31bA
PPase	685.56±15.03C	1,632.94±23.53aB	1,982.97±83.96aA

• Averages followed by equal letters, uppercase letters in the row and lowercase letters in the column, do not differ statistically by Tukey’s test (p>0.05).

The study of the pH effect on the pNPPase and PPase activities of the PIPLC-released alkaline phosphatase present in the SPIPLC fraction (see Figures 1 and 2) revealed that the apparent optimum pH of pNPP and PPi hydrolysis were, respectively, 10.5 and 8.0, both in the epiphysis and diaphysis of tadpoles and in the frogs epiphysis.

Figure 1
Effect of pH on pNPPase activity of alkaline phosphatase released by PIPLC present in the SPIPLC fraction of tadpole epiphysis and diaphysis and frog epiphysis. T.E. = Tadpole Epiphysis; T.D. = Tadpole Diaphysis; F.E. = Frog Epiphysis.

Figure 2
Effect of pH on PPase activity of alkaline phosphatase released by PIPLC present in the SPIPLC fraction of tadpole epiphysis and diaphysis and frog epiphysis. T.E. = Tadpole Epiphysis; T.D. = Tadpole Diaphysis; F.E. = Frog Epiphysis.

The study of the effect of different compounds on the activities of alkaline phosphatase released by PIPLC in the SPIPLC fraction (see Table 4) showed a similar behavior when independently comparing the residual activity of pNPPase and PPase in different bone regions.

Table 4
Study of the effect of different compounds on the pNPPase and PPase activities of PIPLC-released alkaline phosphatase in the SPIPLC fraction.

	Inhibitors
Residual Activity (%)	Control	Levamisole (100 mM)	Theophylline (1 mM)	Zinc (100 µM)	Phosphate (10 mM)		EDTA (100 mM)
Tadpole Epiphysis
pNPPase	100	94.201	84.38	70.46	83.60		32.33
PPase	100	89.80	69.80	104.082	---3		23.67
Tadpole Diaphysis
pNPPase	100	94.271	83.61	69.54	83.37		34.41
PPase	100	87.50	71.47	109.292	---3		21.47
Frog Epiphysis
pNPPase	100	97.481	87.30	72.88	88.04		33.68
PPase	100	85.63	70.99	100.282	---3		25.35

• ¹
  In the case of Levamisole (100 mM), there was no inhibition of the pNPPase activity of PIPLC-released alkaline phosphatase, since only values above 5% were considered inhibitory;
• ²
  In the case of Zinc (100 µM), the value higher than the Control value presented by PPase activity is not considered an activity induction because it is a subtle increase;
• ³
  The activity of PPase could not be measured in the presence of Phosphate (10 mM) since the methodology of determination of this enzymatic activity is performed by the Phosphate Measuring released in solution after the enzyme activity.

The study of the effect of the concentration of pNPP and PPi substrates on the activity of alkaline phosphatase released by PIPLC was carried out in the SPIPLC fraction of tadpoles’ epiphysis and diaphysis and frogs’ epiphysis (see Table 5). V_max values (see Table 5) for pNPPase and PPase differed, but followed the same pattern when comparing the different bone regions, being higher in frog epiphyses, followed by tadpole diaphysis and being lower in tadpole epiphysis.

Table 5
Kinetic parameters for the activities (U.mg^-1±standard error of average) of pNPPase and PPase of alkaline phosphatase present in the SPIPLC fraction.

Kinetic parameters
	Vmax	Km	nH	Vmax/Km
Tadpole Epiphysis
pNPPase	800.20±2.82	0.63±0.009	1.04±0.012	1,269.76
PPase	704.60±14.77	0.01±0.001	1.45±0.15	73,480.03
Tadpole Diaphysis
pNPPase	1,157.00±8.40	0.59±0.017	1.04±0.027	1,960.68
PPase	2,093.00±57.47	0.07±0.008	1.10±0.12	28,916.83
Frog Epiphysis
pNPPase	1,885.00±9.54	0.60±0.012	1.01±0.019	3,160.63
PPase	2,248.00±96.32	0.11±0.017	1.00±0.16	20,911.63

• V_max = maximum speed; K_m = Michaelis-Menten constant; n_H = Hill coefficient. The analyses were performed in GraphPad Prism version 5.01, to estimate the values of V_max and K_m the mathematical model Y=V_max*X/(K_m+X) was used. To calculate the Hill coefficient we used the model Y=V_max*X^h/(K_prime+X^h).

4. Discussion

The activities of alkaline, acid and acid tartrate-resistant phosphatases were studied in the different anatomical regions of the femur, diaphysis and epiphysis, and in the different life stages of the animal, larval and adult (see Table 1), to follow and understand the ossification processes from the beginning of formation to the growth in thickness and length of the bones of members of the Lithobates catesbeianus species.

The pNPPase and PPase activities of alkaline phosphatase in all protein purification fractions present numerically higher values than the activities of acid and acid tartrate-resistant phosphatases in both the epiphysis and diaphysis of tadpoles (see Table 1). Alkaline phosphatase is a biochemical marker of osteoblasts, whereas tartrate-resistant acid phosphatase is considered a biochemical marker of osteoclasts (Gijsbers et al., 2001; Johnson et al., 2000; Pizauro Junior et al., 2017). It can be said that, at this stage, there is greater bone growth than resorption, both in thickness and length, since the limbs of the animal are at the beginning of its development (Felisbino and Carvalho, 1999, 2001; Junqueira and Carneiro, 2013).

Alkaline phosphatase activity is higher in the diaphysis than in the tadpole epiphysis, while acid phosphatases activity is higher in the epiphysis than in the diaphysis (as shown in Table 1), indicating that bones begin to form at the center, following towards the ends (Çiçek et al., 2011; Felisbino and Carvalho, 1999, 2001; Florencio-Silva et al., 2015; Fröbisch, 2008; Gómez et al., 2017; Junqueira and Carneiro, 2013; Long and Ornitz, 2013; Pizauro Junior et al., 2002, 2017; Song et al., 2010; Wu et al., 2016), enabling formation, and growth in thickness and length by the two types of ossification necessary for the animal's transition from the aquatic to the terrestrial environment (Bo et al., 2018; Gao et al., 2018; Nakajima and Yaoita, 2003).

In the case of frogs (see Table 1), we can see that, in the epiphysis, enzyme activities follow the same pattern found in tadpoles, with alkaline phosphatase activity being higher than those of acid phosphatases. In the diaphysis, the values of acid phosphatases activity and the pNPPase activity of alkaline phosphatase are low in fractions CES and S1, and were not detected in fractions P1, SPIPLC, and PPIPLC. In the case of PPase, no activity was detected in any of the fractions (as shown in Table 1).

The profile of the enzymatic activities in the two frog bone regions (see Table 1) shows that, by adulthood, the trabecular bones are already formed and are rigid enough to allow the animal to move through jumps in the terrestrial environment (Bo et al., 2018; Fabrezi et al., 2017; Gao et al., 2018; Pough el al., 2008). Therefore, the longitudinal growth of the long bones still needs to occur, in addition to the maintenance of its diameter, allowing the growth of the animal, as well as the increase of its body mass (Felisbino and Carvalho, 1999, 2001; Junqueira and Carneiro, 2013). In this sense, studies have shown that in already mineralized regions the alkaline phosphatase activities tend to decrease (McWhinnie et al., 1971), which can also be observed in the present work.

The study of the effect of pH on pNPPase activity of alkaline phosphatase present in the SPIPLC fraction (see Figure 1) revealed similar results to those observed for alkaline phosphatase from other sources (McComb et al., 1979), as in Rana pipiens bones, with a pH=10.4 (McWhinnie et al., 1971), in rat bones with a pH=9.4 (Pizauro Junior et al., 1995), in artificial EMVs with a pH=10.0 (Simão et al., 2010), and in tadpoles tails of Lithobates catesbeianus, with a pH=10.5 (Gonçalves et al., 2015).

The study of the effect of pH on the enzymatic PPase activity of alkaline phosphatase present in the SPIPLC fraction (see Figure 2) revealed results similar to those obtained in research using the same substrate, as in studies with rat bones, showing an apparent optimal pH of 8.0 (Pizauro Junior et al., 1995; Rezende et al., 1998) and studies with artificial EMVs with a pH value of 9.0 (Simão et al., 2010).

The study of inhibitors makes it possible to investigate the mechanism of action and regulation of enzymatic activity, and contribute to the understanding of the role of metal ions in the maintenance, native configuration of the protein molecule, its stability, enzymatic activity and the presence of isoenzymes. In this regard, several alkaline phosphatase inhibitors are known and widely used for enzymatic characterization.

In the presence of Levamisole (100 mM) there was no inhibition (approximately 5%) in the pNPPase activity of alkaline phosphatase of tadpole and frog bones (see Table 4), contrarily to what was shown by Simão et al. (2007) in their studies with ectopic ossification and bone marrow of rats, in which this compound was one that most inhibited this enzymatic activity. In the case of PPase activity, there was an inhibition of approximately 12%, suggesting that substrate binding to the enzyme catalytic site did not lead to conformational change that exposed the inhibitor binding site (Pizauro Junior et al., 1995).

Theophylline is a structural amino acid analogue and may occupy a binding site that can be exposed when the enzyme reacts with the substrate (Farley et al., 1980). In the presence of this compound (1 mM), inhibition of pNPPase activity was approximately 15% (see Table 4), relatively low compared to the values obtained in other studies (Gonçalves, 2017; Simão et al., 2007). For PPase activity, theophylline showed an inhibition of approximately 30%.

Regarding the approximate 30% inhibition by zinc (100 µM) in pNPPase activity (see Table 4), it has been proposed that this ion acts by displacing magnesium from its catalytic site, inhibiting the alkaline phosphatase activity of bone tissue (Pizauro Junior et al., 1998). However, in the study of PPase activity, performed in the absence of magnesium, there was no inhibition, suggesting that the true substrate of the enzyme is PPi and not the PPi-Mg complex (Leone et al., 1998).

In the case of inorganic phosphate (10 mM), there was approximately a 15% inhibition of pNPPase activity (see Table 4), although its concentration plays an important role in regulating the ossification process (Millán, 2013). In this sense, it has been found that alkaline phosphatase from different sources is competitively inhibited by inorganic phosphate, one of the reaction products (Pizauro Junior et al., 1988). It was not possible to perform the experiment to analyze the inhibition of this compound on PPase activity, because the methodology used when using this substrate measures the amount of inorganic phosphate released in the reaction medium (Heinonen and Lahti, 1981).

The greatest inhibition of enzymatic activity occurred in the presence of EDTA (100 mM), being approximately 67% for pNPPase activity and 77% for PPase activity (see Table 4), suggesting that alkaline phosphatase from tadpoles and frogs bones of Lithobates catesbeianus is a metalloenzyme (Sharma et al., 2014), as proposed by Pizauro Junior et al. (1998) in their studies with rat bone plate, or that the compound causes conformational changes through interactions with the molecule (Curti et al., 1987).

The V_max value in the SPIPLC fraction was 800.20, 1,157.00, and 1,885.00 U.mg^-1 in the tadpole epiphysis, the tadpole diaphysis, and the frog epiphysis, respectively (see Table 5), while the V_max values of PPase activity were 704.60, 2,093.00, and 2,248.00 U.mg^-1 in the tadpole epiphysis, the tadpole diaphysis, and the frog epiphysis, respectively (see Table 5). These results demonstrate that, weather the alkaline phosphatase released by PIPLC use the chromogenic (pNPP) or physiological (PPi) substrate, the higher activity is due to the greater formation of bone tissue in the frog epiphysis, probably due to the size of the animal. Regarding the tadpole, the largest formation of bone tissue occurs in the diaphysis in relation to the epiphysis. In their studies with Rana catesbeiana (Lithobates catesbeianus), Felisbino and Carvalho (1999, 2001), propose that, in the larval phase, periosteal ossification is mainly responsible for growth in both diameter and length, and that in adulthood, periosteal ossification also remains primarily responsible for the growth and formation of frog limb bones, with only a small amount formed by endochondral ossification, partly responsible for the longitudinal growth of the bone tissue.

Similar apparent optimal pH (see Figures 1 and 2) and K_m (see Table 5) values for each substrate, as well as a similar behavior of residual enzymatic activity in the presence of different compounds (see Table 4), suggests that regardless of the type of ossification occurring, the same enzyme or isoenzymes act (Sharma, Pal and Prasad, 2014) during the process in both bone regions and in the different life stages of the animal.

Statistical differences (see Tables 2 and 3) and the pattern of V_max values (see Table 5) of pNPPase and PPase activities confirm the pattern of bone formation and growth from the center to the extremities, because where there is a higher activity of alkaline phosphatase, more ossification occurs (Çiçek et al., 2011; Felisbino and Carvalho, 1999, 2001; Fröbisch, 2008; Gómez et al., 2017; Junqueira and Carneiro, 2013; Pizauro Junior et al., 1995, 2017; Song et al., 2010).

Comparing the apparent optimal pH (see Figures 1 and 2), K_m and V_max/K_m (see Table 5) values of pNPPase and PPase activities, we can suggest that PPi is the physiological substrate of alkaline phosphatase (Millán, 2006; Rezende et al., 1998), because its apparent optimal pH (pH=8.0) is closer to the physiological one, the lower values of Km indicate a higher affinity of the enzyme for PPi, and the values of V_max/K_m indicate a greater efficiency of enzyme activity when this substrate is used.

Regarding the values of n_H (Hill coefficient) (see Table 5), it is possible to state that, among the studied samples, no site-site interaction was observed, which indicates a “Michaelian” behavior of alkaline phosphatase (Pizauro Junior et al., 1995) in the presence of the two substrates used, therefore, we can use the chromogenic artificial substrate (pNPP) in vitro to study the behavior of this enzyme, relating to the presence of its physiological substrate (PPi) in vivo.

The present study shows that alkaline phosphatase released from the membrane by the action of B. cereus PIPLC presents the same kinetic properties in relation to the optimum pH of action, affinity for substrates, and the action of compounds in different bone regions, regardless of the type of bone ossification that is occurring, and in the different phases of the animal's life, both aquatic and terrestrial.

The data show that phosphatases activities are higher in frog epiphysis, when compared to tadpole bone regions. This effect of higher activity during pH studies is also observed on the other experiments. Although the specific activity is higher, the kinetic parameters are the same. With pH analysis we can see that despite the bone region or development stage (tadpole or frog) the same apparent optimum pH is observed.

The results of this work are similar to those of studies with other vertebrates, which may be indicative of the conservation of the genes of the enzymes studied during the evolutionary process, demonstrating the potential of amphibians as excellent animal models for the study of the biological mineralization process. In this regard, more studies involving gene expression of bone markers and enzymatic activity with different animal models may be important to corroborate the hypothesis that amphibians may be used as a broad ossification model.

Supplementary Material

Supplementary material accompanies this paper.

Supplementary Figure 1 Scheme of the purification of membrane-bound proteins by phosphatidylinositol anchor and the obtaining of enzymatic extracts.

This material is available as part of the online article from https://www.scielo.br/j/BJB

Acknowledgements

This work was supported by grants from FAPESP, CNPq, and CAPES.

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Publication Dates

• Publication in this collection
  25 Oct 2021
• Date of issue
  2024

History

• Received
  07 May 2021
• Accepted
  15 July 2021