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

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

doi:10.1590/1519-6984.251970

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Original Article • Braz. J. Biol. 84 • 2024 • https://doi.org/10.1590/1519-6984.251970 copy

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

Authorship SCIMAGO INSTITUTIONS RANKINGS

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

Regions/Fractions	Protein (mg/mL)	pNPPase		PPase		Ac.P.		T.R.Ac.P.
Regions/Fractions	Protein (mg/mL)	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

Factors			Specific activity (nmol/min/mg)
Enzymatic activities	Bone regions		Specific activity (nmol/min/mg)
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^ significant at the 1% probability level; VC% = variation coefficient.
Bone regions		<0.001**
Enzymes X Bone regions		<0.001**
VC%		3.07%

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

	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 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;	87.30	72.88	88.04	33.68
PPase	100	85.63	70.99	100.28² 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 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.	25.35

Kinetic parameters
	V_max	K_m	n_H	V_max/K_m
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

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