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

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.

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 Km 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.

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 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.

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 Km 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.

Ativação de células de memória na produção de anticorpos e na expressão de células IgM positivas no baço de tilápias-do-nilo / Activation of memory cells in the antibody production and the expression of IgM positive cells in Nile tilapia spleen

O presente trabalho avaliou o papel do baço no armazenamento e na reativação das linhagens de células B, representadas por células IgM positivas imunomarcadas no tecido esplênico, bem como a funcionalidade dessas células, sobre a cinética dos linfócitos e na produção sistêmica de anticorpos em tilápias-do-nilo (Oreochromis niloticus). Foram separados dois grupos: grupo memória, constituído por peixes previamente imunizados com hemácia de carneiro a 2,5%, para a geração da memória imune, e o grupo naive, que recebeu o mesmo volume de solução salina a 0,65%. Após 32 dias, os dois grupos foram submetidos a uma nova dose do antígeno na mesma concentração, volume e via de inoculação. A reativação dos clones de memória foi evidenciada pelo aumento do número de células IgM positivas no baço do grupo memória no dia zero/pré-imune. Além disso, o mesmo grupo apresentou aumento dos títulos de anticorpos séricos no 14º dia e no número absoluto de linfócitos no 21º dia em relação ao grupo naive. Esses resultados sugerem que o baço não seja apenas um local de armazenamento, mas também de reativação de células B de memória em tilápia-do-nilo.(AU)

This work aimed to evaluate the role of the spleen in storage and reactivation of the memory B cells, represented by IgM positive cells and the systemic production of sheep antibodies anti-red cell in Nile tilapia (Oreochromis niloticus). Two groups were established: the memory group, containing fish previously immunized with a 2,5% sheep anti-red cell, to generate the immune memory; and the naive group, containing fish that received a 0,65% saline solution. After 32 days, both groups were subjected to a new dose of the same antigen at the same concentration, volume, and inoculation via. The memory clones reactivation was correlated to the increase of the IgM positive cells in the spleen in the memory group at 0 day. The memory group showed an increase in the absolute number of lymphocytes at 21 days and an increase in the antibodies at 14 days after inoculation when compared to the naive group. The results suggest that the spleen may be a storage and reactivation place of memory B cells in Nile tilapia.(AU)

Interference of age and supplementation of direct-fed microbial and essential oil in the activity of digestive enzymes and expression of genes related to transport and digestion of carbohydrates and proteins in the small intestine of broilers.

The objectives of this study were to describe alterations that age and dietary inclusion of direct-fed microbial (DFM) Bacillus subtilis (BS) and a specific essential oil (EO) blend (carvacrol, cinnamaldehyde, cineol, and pepper extract) causes in the activity of digestive enzymes (maltase: MALT; aminopeptidase-N: APN; intestinal alkaline phosphate: IAP) and expression patterns of genes related to transport (oligopeptide transporter gene: SLC15A1; Na+-dependent glucose and galactose transporter gene: SLC5A1; Na+-independent glucose, galactose, and fructose transporter gene: SLC2A2; ATPase, Na+/K+ transporting gene: ATP1A1) and digestion (aminopeptidase-N gene: ANPEP; maltase-glucoamylase gene: MGAM; Sucrase-isomaltase gene: SI) of carbohydrates and proteins in the small intestine of broilers. Also, the objective was to analyze if growth performance of broilers is affected by supplementation (BS and EO blend). Day-old male broiler chicks (n = 1,320) were assigned to 5 treatments. Diets included a basal diet (BD) as a negative control (CON); experimental diets were BD + BS; BD + BS + EO; BD + EO; BD + antibiotic growth promoter (AGP) avilamycin was the positive control. Performance was evaluated between 1 to 42 d. Transcript abundance of transport-related genes and digestion-related genes were assayed by RT-qPCR and determined at d 7, 21, and 42. MALT-, APN-, and IAP-specific activities were determined at d 7, 21, and 42. Broilers fed BS had greater SLC15A1 mRNA abundance compared to CON, while EO and AGP were related to higher activities of IAP and APN. Analysis over time revealed higher abundance of MGAM, SLC2A2, SLC15A1, SLC5A1 and SI mRNA at d 42 when compared to d 7. Activity of IAP decreased after d 7 and activity of MALT increased with age. The current study suggests that age had effect over carbohydrate and protein transport and carbohydrate digestion. The supplementation of BS DFM hade evident effect over protein transport and that the use of EO in the diet enhanced the activities of carbohydrate and protein digestion, reflecting improvement in digestive and transport physiology of birds. Changes performed by BS DFM and EO did not favor performance.

Breeder age and bone development in broiler chicken embryos / Idade da matriz e desenvolvimento ósseo em embriões de frango de corte

The effect of breeder age on long bone development was studied in chicken embryos from 12 days of incubation until hatching. Fertile eggs were incubated and randomly assigned in a 2 x 6 factorial arrangement (two breeder ages - 38 and 60 weeks and six incubation days - 12, 14, 16, 18, 20, and 21). Enzymatic activity of acid and alkaline phosphatases in tibial epiphyses and weights as well as length and width in tibias and femurs of the embryos were determined. Tartrate-resistant acid and alkaline phosphatases activity in epiphyses was not affected by breeder age. Absolute weight and width of femur and tibia were larger in 60-week-old embryos compared to 38-week-old. Enzymatic activity and morphometric measurements increased with incubation day, independently of breeder age. The results showed that the process of endochondral ossification during the last two thirds of embryo development was not influenced by the age of the breeders. Although, in terms of absolute weight, the long bones of embryos from older breeders were heavier, which was associated with the larger width of the bones, but and not with their length.

O efeito da idade da matriz sobre o desenvolvimento dos ossos longos foi estudado em embriões de frango de 12 dias de incubação até o nascimento. Ovos férteis foram incubados e distribuídos em delineamento inteiramente casualizado em arranjo fatorial 2 x 6 (duas idades de matriz - 38 e 60 semanas e seis dias de incubação - 12, 14, 16, 18, 20 e 21 dias). Determinou-se a atividade enzimática das fosfatase alcalina e ácida-resistente ao tartrato no peso e nas epífises da tíbia, no comprimento e na largura da tíbia e do fêmur. A atividade das fosfatases não foi afetada pela idade da matriz. O peso absoluto e a largura de fêmur e tíbia foram maiores nos embriões das matrizes com 60 semanas de idade. Atividade enzimática e medidas morfométricas aumentaram com o dia de incubação independentemente da idade da matriz. Concluiu-se que o processo de ossificação endocondral durante os dois últimos terços de desenvolvimento embrionário não foi influenciado pela idade das matrizes. No entanto, em termos de peso absoluto, os ossos longos de embriões provenientes de matrizes velhas foram mais pesados o que foi associado à maior largura e não ao maior comprimento dos ossos.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugar cane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80 similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme

Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochodral ossification.

Treatment with phosphatidylinositol-specific phospholipase C of rat osseous plate membranes released up to 90-95% of alkaline phosphatase, but a specific ATPase activity (optimum pH = 7.5) remained bound to the membrane. The hydrolysis of ATP by this ATPase was negligible in the absence of magnesium or calcium ions. However, at millimolar concentrations of magnesium and calcium ions, the membrane-specific ATPase activity increased to about 560-600 U/mg, exhibiting two classes of ATP-hydrolysing sites, and site-site interactions. GTP, UTP, ITP, and CTP were also hydrolyzed by the membrane-specific ATPase. Oligomycin, ouabain, bafilomycin A1, thapsigargin, omeprazole, ethacrynic acid and EDTA slightly affected membrane-specific ATPase activity, while vanadate produced a 18% inhibition. The membrane-specific ATPase activity was insensitive to theophylline, but was inhibited 40% by levamisole. These data suggested that the membrane-specific ATPase activity present in osseous plate membranes, and alkaline phosphatase, were different proteins.

Conidial alkaline phosphatase from Neurospora crassa.

An alkaline phosphatase was purified from conidia of a Neurospora crassa wild type strain. The M(r) of the purified native enzyme was estimated as ca 145,000 and 110,000 by gel filtration, in the presence and absence of magnesium ions, respectively. A single polypeptide band of M(r) 36,000 was detected by SDS-PAGE, suggesting that the native enzyme was a tetramer of apparently identical subunits. Conidial alkaline phosphatase was an acidic protein (pl = 4.0 +/- 0.1), with 40% carbohydrate content. Optimal pH was affected by substrate concentration and magnesium ions. Low concentrations of calcium ions (0.1 mM) had slight stimulatory effects, but in excess (5 mM) caused protein aggregates with decreased activity. The enzyme specificity against different substrates was compared with those reported for constitutive or Pi-repressible alkaline phosphatases produced by N. crassa. The results suggested that the conidial alkaline phosphatase represented a different class among other such enzymes synthesized by this organism.

Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate.

Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process.

Allosteric modulation by ATP, calcium and magnesium ions of rat osseous plate alkaline phosphatase.

Alkaline phosphatase from rat osseous plate is allosterically modulated by ATP, calcium and magnesium at pH 7.5. At pH 9.4, the hydrolysis of ATP and PNPP follows Michaelis-Menten kinetics with K0.5 values of 154 microM and 42 microM, respectively. However, at pH 7.5 both substrates exhibit more complex saturation curves, while only ATP exhibited site-site interactions. Ca(2+)-ATP and Mg(2+)-ATP were effective substrates for the enzyme, while the specific activity of the enzyme for the hydrolysis of ATP at pH 7.5 was 800-900 U/mg and was independent of the ion species. ATP, but not PNPP, was hydrolyzed slowly in the absence of metal ions with a specific activity of 140 U/mg. These data demonstrate that in vitro and at pH 7.5 rat osseous plate alkaline phosphatase is an active calcium or magnesium-activated ATPase.

Polyoxyethylene 9-lauryl ether-solubilized alkaline phosphatase: synergistic stimulation by zinc and magnesium ions.

1. Polidocanol-solubilized apoalkaline phosphatase could be stimulated either by zinc ions (Kd = 8.5 nM) or by magnesium ions alone (Kd = 3.8 microM). 2. Zinc and magnesium ions had synergistic effects on Polidocanol-solubilized apoalkaline phosphatase, leading to a fully active enzyme (700-800 U/mg). 3. Zinc ions inhibited non-competitively the Polidocanol-solubilized apoenzyme (Ki = 7.1 microM) by displacing magnesium ions from their binding sites. 4. A model for the action of zinc and magnesium ions on the modulation of the enzyme activity is proposed.

Solubilization of membrane-bound matrix-induced alkaline phosphatase with polyoxyethylene 9-lauryl ether (polidocanol): purification and metalloenzyme properties.

1. Matrix-induced alkaline phosphatase prepared from rat osseous plate was solubilized with polidocanol and purified on a Sephacryl S-300 column. 2. Purified solubilized alkaline phosphatase has a molecular weight of ca 115,000 and bind one magnesium and two zinc ions. At least 110 detergent molecules are bound to each enzyme molecule. 3. Solubilization and purification procedures did not destroy the ability of the enzyme to hydrolyze adenosine-5'-triphosphate, p-nitrophenylphosphate, pyrophosphate and bis p-nitrophenylphosphate. 4. Magnesium, manganese and cobalt ions are stimulators of PNPPase activity of solubilized enzyme whereas calcium and zinc ions are inhibitors.