Alteration of adenoid tissue alkaline and acid phosphatase in children with secretory otitis media.

OBJECTIVE: The role of pharyngeal lymphoid tissue in etiopathogenesis of secretory otitis is not yet defined. The influence of tonsillar and adenoid mass, weight, obstruction of naspharyngeal orrifitium, bacterial reservoire or some immunological events are of scientific interest. Tissue nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) are enzymes detected in lymphoid tissue, TNAP as characteristic of B cells, ACP as a characteristic of macrophages and folucullardentritic cells. These enzymes interfere in cell metabolism by removing 5' phosphate group from nucleotides and proteins. Specific activity and kinetic properties were studied in palatinal tonsils and adenoids of children with secretory otitis (OME) and compared with children with recurrent tonsillitis without ear involvement. METHOD: Adenoid and tonsillar tissue of l7 children with OME and 30 children with recurrent tonsillitis were subjected to biochemical investigation using method of releasing of p-nitrophenol from p-nitrophenylphosphate (pNPP). Kinetic parameters as Michaelis-Menten constant were calculated by non-linear regression estimation method. RESULTS: Specific activity of adenoid alkaline phosphatase was lower in children with OME in relation to children with recurrent tonsillitis (t=5.733507, p<0.01). Specific activity of adenoid acid phosphatase was also lower in children with OME (t=3.655456, p<0.01). pH optimum for both enzymes was the same in these two groups of children. Michaelis-Menten constant for both enzymes was significantly higher in adenoid of children with OME than in children with recurrent tonsillitis suggesting lower enzyme affinity for the substrate. CONCLUSION: Differences in specific activities and kinetic properties of adenoid alkaline and acid phosphatases between children with OME and children with recurrent tonsillitis without OME were verified in this study. The results of the study are not able to explain the alteration of alkaline and acid phosphatase characteristics but could point to some possible and specific role of nasopharyngeal lymphoid tissue in pathogenesis of secretary otitis.

Parótida bovina: purificaçäo e caracterizaçäo da fosfatase ácida de baixo peso molecular / Bovine kidney: purification and characterization of acid phosphatase of low molecular weight

A fosfatase ácida de BPM da parótida bovina foi purificada 1.800 vezes até a homogeneidade, com rendimento de 8 por cento, através de um procedimento envolvendo fracionamento com sulfato de amônio, tratamento ácido e cromatografia de troca iônica em SP-Sephadex com eluiçäo por íon-afinidade. Os critérios de pureza utilizados foram a A.E., PAGE, SDS-PAGE, filtraçäo em gel (Superdex HR 70) e análise da composiçäo de aminoácidos. A enzima purificada (A.E. de 100 µmol min-1 mg-1) é composta por uma cadeia polipeptídica simples e possui Mr de 13,6 e 19 kDa, como determinado através da filtraçäo em gel e SDS-PAGE, respectivamente. A composiçäo parcial de aminoácidos (Cys e Trp näo foram determinados) foi obtida após a hidrólise ácida da proteína purificada seguida da análise dos resíduos de aminoácidos e evidenciou a existência de, pelo menos, 151 resíduos de aminoácidos...

Does gene palB regulate the transcription or the post-translational modification of Pi-repressible phosphatases of Aspergillus nidulans?

When grown on low-Pi medium, the chaA1 pabaA1 palB7 mutant of Aspergillus nidulans excretes an acid phosphatase with steady-state kinetic properties, temperature sensitivity and electrophoretic mobility different from those of the enzyme excreted by the pabaA1 strain. The enzyme excreted by the pabaA1 strain at pH 6.5 showed PNP-P activity with negative cooperativity (K0.5 = 0.87 + or - 0.06 mM, n = 0.68 + or - 0.03) whereas the enzyme excreted by the chaA1 pabaA1 palB7 mutant showed Michaelian kinetics (Km = 0.46 + or - 0.03 mM, n = 1.00 + or - 0.02). The apparent half-lives at 60§C, pH 5.5, of acid phosphatase excreted by the pabaA1 and chaA1 pabaA1 palB7 strains were 58.6 + or - 4.9 min and 21.5 + or - 1.8 min, respectively. Furthermore, the electrophoretic mobility of acid phosphatases excreted by the palA1, palB7, palC4, palE11 and palF15 mutants of A. nidulans was altered and differed from the electrophoretic mobility of the enzyme excreted by the wild-type strain. Also, the palB7 mutation altered the electrophoretic pattern of acid phosphatases synthesized on high-Pi medium. These results are compatible with the post-translational modifications in the Pi-repressible phosphatases rather than with the action of gene palB in controlling the transcription of structural genes of these enzymes

Endocytosis of lysosomal acid phosphatase; involvement of mannose receptor and effect of lectins.

Acid phosphatase and beta-glucosidase are unique among lysosomal enzymes in that they have both high mannose and complex type sugasr chains, whereas oligosaccharide chains of lysosomal enzymes in matrix are of high mannose type. We have previously shown that beta-glucosidase was endocytosed into macrophages via an unidentified receptor different from a mannose/fucose receptor (K. Imai, Cell Struct. Funct. 13, 325-332, 1988). Here, we show that uptake of acid phosphatase purified from rat liver lysosomes into rat macrophages was inhibited by ligands for a mannose/fucose receptor and was mediated via an apparently single binding site with Kuptake of 24.7 nM. These results indicate that acid phosphatase and beta-glucosidase recognize different types of receptors even if they have similar sugar chains. Polyvalent concanavalin A which binds both to the enzyme and to macrophages specifically stimulated the uptake in a dose dependent manner, whereas wheat germ agglutinin and phytohaemagglutinin did not.

Phosphorylation of Escherichia coli enolase.

In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography. Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue. Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine. In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase. When specific radioactivities of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells. At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate. When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed. Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction. Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.