Eulázio Mikio Taga

Purification and characterization of multiple forms of soybean seed acid phosphatases

Abstract Four isoforms of acid phosphatase (EC 3.1.3.2), AP1, AP2, AP3A and AP3B, have been detected and partially purified from soybean seed ( Glycine max ) through DEAE- and SP-Sephadex chromatographies. Specific activity values of 822, 163, 14 and 66 nkat·mg −1 were obtained for AP1 (903-fold purification), AP2 (180-fold), AP3A (15-fold), and AP3B (73-fold), respectively, using p -nitrophenylphosphate as substrate. Relative native molecular mass values for AP1. AP2, AP3A and AP3B, determined by gel filtration on calibrated SW-300 Waters Protein Glass column, were found to be 51 000, 58 000, 52 000 and 30 000, respectively. All four acid phosphatase isoforms presented a carbohydrate moiety in their structures and revealed only single phosphatase activity bands following nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. AP1 and AP2 exhibited greater substrate specificity than AP3A and AP3B. The K m values were determined for p -nitrophenylphosphate, tyrosinephosphate and inorganic pyrophosphate, at pH 5.0 and 37 °C. The acid phosphatases presented the following apparent K m values: AP1 ( p NPP — 0.49, PPi — 0.21 and TyrP — 1.14 mM); AP2 ( p NPP — 0.38, PPi — 1.33 and TyrP — 1.14 mM); AP3A ( p NPP -0.20, PPi — 0.16 and TyrP — 0.19 mM) and AP3B ( p NPP — 0.086, PPi — 0.17 and TyrP — 0.17 mM). All four isoforms were inhibited by inorganic phosphate, fluoride, vanadate, molybdate, Cu 2+ and Zn 2+ . The soybean seed acid phosphatases did not catalyze the transphosphorylation reaction since no stimulation was observed with inorganic phosphate acceptors, such as glycerol, methanol and ethanol.

Bovine kidney low molecular weight acid phosphatase: FMN‐dependent kinetics

A low molecular weight bovine kidney acid phosphatase, electrophoretically homogeneous and with a relative molecular mass of 17.8 kDa, was used in this work. Among the various substrates tested, FMN was found to be the most effective, at pH 7.0. Distinct activation energy values were obtained for p‐nitrophenyl phosphate‐ (45.44 kJ mol‐1) and flavin mononucleotide‐ (28.60 kJ mol‐1) hydrolysis reactions. The FMN hydrolysis was strongly inhibited by Cu2+ and pCMB, but activated by guanosine. Pyridoxal‐phosphate and vanadate were competitive inhibitors for the FMN‐dependent reaction.

Tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats

The treatment of persistent bone defects has encouraged the search for proper techniques or bone substitutes. In Dentistry, a common problem in the treatment of periodontal bone defects is the growth of tissues within the lesion, such as the junctional epithelium, which impair regeneration of these tissues. Guided tissue regeneration (GTR), based on the separation of the tissues by means of membranes or barriers, was developed in an attempt to improve periodontal regeneration. The aim of this study was to histologically evaluate the tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats. The study periods were 1, 3, 7, 15, 30 and 60 days after implantation. Analysis of the histological sections demonstrated a moderate to intense inflammatory response at 1 and 3 days, moderate at 7 and 15 days, and almost absent at 30 and 60 days. Resorption of the membrane began 15 days after implantation, and at 60 days only remnants could be detected in some animals. We concluded that the demineralized bovine cortical bone membrane was well tolerated by the tissues and is completely resorbed after 30-60 days by mononuclear cells and multinucleated giant cells, which disappear upon completion of the process.

Biocompatibility of EDTA, EGTA and citric acid

This in vivo study evaluated, through the physicochemical assay method for quantification of enhanced vascular permeability, the irritating potential of EDTA, EGTA, citric acid and saline. Thirty-two male Wister rats were anesthetized and four experimental sites were demarcated on their backs. Injections of 2% Evans blue (20 mg/kg) were administered intravenously into the lateral caudal vein. The test solutions were immediately injected intradermally (0.01 mL) into the experimental sites. The animals were killed 30 min, 1, 3 and 6 h after injection of the solutions and each piece of skin was submerged in formamide and incubated at 45 masculineC for 72 h. After filtration, the optical density was measured in a spectrophotometer and the total amount of dye extracted from the samples was calculated by means of a standard calibration curve. Data were analyzed statistically by two-way ANOVA and Tukeys HSD test. Compared to control, EDTA had the greatest volume of dye followed by EGTA and citric acid, for all time periods. There were statistically significant differences between all solutions (p<0.01). Considering the periods assessed, a significant difference was observed between the 3- and 6-h groups (p<0.05), but not between the 30-min and 1-h groups. Among the organic acids evaluated in this study, citric acid yielded the lowest amount of extracted dye. This indicates that the citric acid was the least irritating solution.

Endogenous lectin as a possible regulator of the hydrolysis of physiological substrates by soybean seed acid phosphatase

The effects of two lectins concanavalin A (conA) and soybean agglutinin, on soybean seed acid phosphatase activity were investigated using p-nitrophenylphosphate (pNPP), pyrophosphate (PPi) and phosphoenolpyruvate (PEP) as substrates. Of the four acid phosphatase isoforms (AP1, AP2, AP3A and AP3B) purified from soybean seeds, only AP1 was activated 40 and 60% by conA and soybean agglutinin, respectively. Both lectins affected some of the kinetic parameters of AP1. The activation by lectins was not affected by 1 mM Ca2+ or Mn2+ but glucose and methylmannopyranoside (100 mM) prevented activation by conA. Under the same conditions, galactose had no effect. These results suggest that plant acid phosphatases may be regulated by lectins, the effects vary according to the substrate used.

Glycolytic intermediates as substrates of soybean acid phosphatase isoforms

Abstract The kinetic properties of four isoforms of acid phosphatase purified from mature soybean ( Glycine max ) seeds were studied using glycolytic metabolites as substrates. The isoforms AP1, AP3A and AP3B presented maximal activities around pH 4.0, and AP2 at pH 6.0, for phosphoenolpyruvate (PEP) as a substrate. With glucose-6-P (G6P) and fructose-6-P (F6P) maximal activities were observed at pH 5.5 for the AP3A and AP3B isoforms. The acid phosphatases presented the following apparent K m values, at the corresponding optimum pH values: AP1 ( p -nitrophenylphosphate (pNPP): 0.49, PEP: 0.23 mM); AP2 (pNPP: 0.38, PEP: 0.47 mM); AP3A (pNPP: 0.20, PEP: 0.10, G6P: 0.30, F6P: 0.16 mM) and AP3B (pNPP: 0.086, PEP: 0.078, G6P: 0.31, F6P: 0.33 mM). Maximal specificity constant ( V max / K m ) was obtained for the isoform AP1, with PEP as a substrate. Independent of the substrates, the reactions catalyzed by the soybean acid phosphatase isoforms were potently inhibited by molybdate and to a lesser extent by Zn 2+ . Inhibitions were also observed in the presence of fluoride, with PEP as a substrate, and by Cu 2+ , and p -chloromercuribenzoate (pCMB) with G6P and F6P as substrates. Our results suggest that the four acid phosphatase forms could play important roles in plant metabolism, acting on key glycolytic intermediates.

Inhibition of Bovine Kidney Low Molecular Mass Phosphotyrosine Protein Phosphatase by Uric Acid

Uric acid inhibited 50% of the activity of bovine kidney low molecular mass phosphotyrosine protein phosphatase at concentrations of 1.0, 0.4, 1.3, and 0.2 mM, respectively for p -nitrophenyl phosphate (p -NPP), flavine mononucleotide, β -naphthyl phosphate and tyrosine phosphate (Tyr-P) as substrates. The mixed type inhibition of p -NPP hydrolysis was fully reversible, with K ic and K iu values of 0.4 and 1.1 mM, respectively; the inhibition by uric acid shifted the pH optimum from 5.0 to 6.5. When Tyr-P was the substrate, competitive inhibition was observed with a K i value of 0.05 mM. Inhibition studies by uric acid in the presence of thiol compounds, and preincubation studies in the presence of inorganic phosphate suggest that the interaction of uric acid with the enzyme occurred at the active site, but did not involve SH residues, and that the mechanism of inhibition depended on the structure of the substrates.

Effect of homologous series of n-alkyl sulfates and n-alkyl trimethylammonium bromides on low molecular mass protein tyrosine phosphatase activity

The effect of anionic and cationic surfactants on acid phosphatase denaturation has been extensively studied. Low molecular mass (LMr) protein tyrosine phosphatase (PTP), a key regulatory enzyme involved in many different processes in the cell, was distinctly affected by anionic (homologous series of n-alkyl sulfates (C8-C14)) and cationic (n-alkyl trimethylammonium bromides (C12-C16)) surfactants. At concentrations 10-fold lower critical micellar concentration (cmc) values, the enzyme was completely inactivated in the presence of anionic surfactants, in a process independent of the pH, and dependent on the chain length of the surfactants. Under the same conditions, the effect of cationic surfactants on the enzyme activity was pH-dependent and only at pH 7.0 full inactivation was observed at concentrations 10-fold higher cmc values. In contrast to cationic surfactants the effect of anionic surfactants on the enzyme activity was irreversible and was not affected by the presence of NaCl. Inorganic phosphate, a known competitive inhibitor of PTP, protected the enzyme against inactivation by the surfactants. Our results suggest that the inactivation of the LMr PTP by anionic and cationic surfactants involved both electrostatic and hydrophobic interactions, and that the interactions enzyme-surfactants probably occurred at or near the active site. (Mol Cell Biochem 265: 133–140, 2004)

AVALIAÇÃO IN VITRO DA LIBERAÇÃO DE FLÚOR DE CIMENTOS DE IONÔMERO DE VIDRO E OUTROS MATERIAIS QUE CONTÊM FLÚOR

The aim of this study was to determine the fluoride release from six fluoride-containing materials over 28 days. The results showed that the fluoride release patterns were similar for all glass ionomers tested, i.e., a large initial release was followed by a rapid decline in the amount released within the second day but became relatively stable after seven days. The release from glass ionomer cements was clearly greater than that from a sealant and a composite resin. Vidrion R released the largest amount of fluoride among all tested materials.

Kinetic Characterization of Bovine Lung Low-Molecular-Weight Protein Tyrosine Phosphatase

Protein tyrosine phosphatase is an important class of enzymes that plays an essential role in the cellular proliferation, differentiation, and oncogenesis. In this paper we report characterization of a low-molecular-weight protein tyrosine phosphatase purified from bovine lung. The enzyme activity was essentially independent of metal ions and sensitive to sulfhydryl reagents. Both vanadate and inorganic phosphate are competitive inhibitors, with Ki values of 0.38 microM and 0.28 mM, respectively. Besides p-nitrophenyl phosphate, the enzyme was also able to efficiently hydrolyze tyrosine phosphate, beta-naphthyl phosphate, and flavine mononucleotide.