Acid phosphatase in blood smears of Phrynops geoffroanus (Testudines: Chelidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Electrophoretic protein pattern and acid phosphatase activity in the midgut extracts of Apis mellifera L. (Hymenoptera: Apidae) during metamorphosis

Foram pesquisadas variações no padrão eletroforético das proteínas e da atividade da fosfatase ácida contidas em extratos do intestino médio de Apis mellifera L. durante o último estágio larval e pupação com a finalidade de estabelecer um paralelo entre os resultados e os eventos da metamorfose. Verificou-se maior variedade de bandas protéicas durante o estágio de pré-pupa e menor na pupa de olho marrom. A atividade da fosfatase ácida foi maior durante o último estágio larval e menor na pupa de olho branco. A maior variedade de bandas protéicas na pré-pupa coincide com a histólise do epitélio larval e reconstituição do epitélio pupal, enquanto a menor variabilidade na pupa de olho marrom coincide com o fim da diferenciação do intestino médio. A maior atividade fosfatásica no último estágio larval pode ocorrer em razão da sua função na histólise do epitélio.

MECHANISM OF ACTION OF COBALT IONS ON RAT OSSEOUS PLATE ALKALINE-PHOSPHATASE

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by cobalt ions in a similar way as by magnesium ions. For concentrations up to 1 mu M, the Chelex-treated enzyme was stimulated by cobalt ions, showing K-d = 6.0 mu M, V = 977.5 U/mg, and site-site interactions (n = 2.5). Cobalt-enzyme was highly unstable at 37 degrees C, following a biphasic inactivation process with inactivation constants of about 0.0625 and 0.0015 min(-1). Cobalt ions stimulated the enzyme synergistically in the presence of magnesium ions (K-d = 5.0 mu M; V = 883.0 U/mg) or in the presence of zinc ions (K-d = 75.0 mu M; V = 1102 U/mg). A steady-state kinetic model for the modulation of enzyme activity by cobalt ions is proposed.

Dependence of divalent metal ions on phosphotransferase activity of osseous plate alkaline phosphatase

Kinetic evidence for the role of divalent metal ions in the phosphotransferase activity of polidocanol-solubilized alkaline phosphatase from osseous plate is reported. Ethylenediamine tetreacetate, 1,10-phenanthrolin, and Chelex-100 were used to prepare metal-depleted alkaline phosphatase. Except for Chelex-100, either irreversible inactivation of the enzyme or incomplete removal of metal ions occurred. After Chelex-100 treatment, full hydrolase activity of alkaline phosphatase was recovered upon addition of metal ions. on the other hand, only 20% of transferase activity was restored with 0.1 mu M ZnCl2, in the presence of 1.0 M diethanolamine as phosphate acceptor. In the presence of 0.1 mM MgCl2, the recovery of transferase activity increased to 63%. Independently of the phosphate acceptor used, the transferase activity of the metal-depleted alkaline phosphatase was fully restored by 8 mu M ZnCl2 plus 5 mM MgCl2. In the presence of diethanolamine as phosphate acceptor, manganese, cobalt, and calcium ions did nor stimulate the transferase activity. However, manganese and cobalt-enzyme catalyzed the transfer of phosphate to glycerol and glucose. (C) 1997 Elsevier B.V.

Purification and properties of acid phosphatase (EC 3.1.3.2) secreted by strain 74A of the mould Neurospora crassa

Both P-i-repressible acid phosphatases, IIb (mycelial) and IIc (extracellular), synthesized by Neurospora crassa and purified to apparent homogeneity by 7.5% PAGE, are monomers, are inhibited by 2 mM ZnCl2 and are nonspecifically stimulated by salts. However, the IIc form is activated by p-nitrophenylphosphate (in a negative cooperativity effect with a K-0.5 of 2.5 mM) whereas form IIb shows Michaelis kinetics, with a K-m of 0.5 mM. Thus, since both enzymatic forms may be expressed by the same gene (pho-3), it is possible that post-translational modifications lead to the excretion of an enzymatic form with altered Michaelis kinetics compared with the enzymatic form retained by the mycelium.

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 muM and 42 muM, respectively. However, at pH 7.5 both substrates exhibit more complex saturation curves, while only ATP exhibited site-site interactions. Ca2+-ATP and Mg2+-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.

THE PHO-2A MUTANT OF NEUROSPORA-CRASSA WHICH IS DEFICIENT IN PI-REPRESSIBLE ALKALINE-PHOSPHATASE (EC 3.1.3.1) IS ALSO DEFECTIVE IN PI-REPRESSIBLE ACID-PHOSPHATASE (EC 3.1.3.2)

1. The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively.2. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t1/2 = 4 min at 54-degrees-C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54-degrees-C, pH 5.4).3. The pho-2A mutant of N. crassa secreted a more thermolabile acid phosphatase (t1/2 = 30 min at 50-degrees-C, pH 5.4) than the wild strain (t1/2 of at least 80 min at 50-degrees-C, pH 5.4).4. The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t1/2 = 37 min at 54-degrees-C, pH 5.4) than the wild strain in high Pi medium (t1/2 = 14 min al 54-degrees-C, pH 5.4).5. The pleiotropic nature of the pho-2 locus and its possible involvement in the mechanism of phosphatase secretion by N. crassa are proposed.

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Effect of calcium ions on rat osseous plate alkaline phosphatase activity

Rat osseous plate alkaline phosphatase is a metalloenzyme with two binding sites for Zn2+ (sites I and III) and one for Mg2+ (site II). This enzyme is stimulated synergistically by Zn2+ and Mg2+ (Ciancaglini et al., 1992) and also by Mn2+ (Leone et al., 1995) and Co2+ (Ciancaglini et al., 1995). This study was aimed to investigate the modulation of enzyme activity by Ca2+. In the absence of Zn2+ and Mg2+, Ca2+ had no effects on the activity of Chelex-treated, Polidocanol-solubilized enzyme. However, in the presence of 10 mu M MgCl2, increasing concentration of Ca2+ were inhibitory, suggesting the displacement of Mg2+ from the magnesium-reconstituted enzyme. For calcium-reconstituted enzyme, Zn2+ concentrations Zip to 0.1 mu M were stimulatory, increasing specific activity from 130 U/mg to about 240 U/mg with a K-0.5 = 8.5 nM. Above 0.1 mu M Zn2+ exerted a strong inhibitory effect and concentrations of Ca2+ up to I mM were not enough to counteract this inhibition, indicating that Ca2+ was easily displaced by Zn2+. At fixed concentrations of Ca2+, increasing concentrations of Mg2+ increased the enzyme specific activity from 472 U/mg to about 547 U/mg, but K-0.5 values were significantly affected (from 4.4 mu M to 38.0 mu M). The synergistic effects observed for the activity of Ca2+ plus magnesium-reconstituted enzyme, suggested that these two ions bind to the different sites. A model to explain the effect of Ca2+ on the activity of the enzyme is presented. (C) 1997 Elsevier B.V.

OSSEOUS PLATE ALKALINE-PHOSPHATASE IS ANCHORED BY GPI

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The Mr of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mu mol min(-1) mg(-1)), ATP (42.0 mu mol min(-1) mg(-1)) and pyrophosphate (28.4 mu mol min(-1) mg(-1)). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited ''Michaelian'' kinetics with K-0.5 = 70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K-d = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (K-d = 6.2 mu M) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K-0.5 = 10.1 mu M), magnesium (K-0.5 = 29.5 mu M) and manganese ions (K-0.5 = 5 mu M) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K-0.5 = 653 mu M) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.

Acid phosphatase activity in Malpighian tubules of Triatoma infestans Klug

Acid phosphatase activity was detected in the Malpighian tubules of the bloodsucking hemipteran, Triatoma infestans. The enzyme activity was especially prominent in the cytoplasmic globules which were assumed to be laminated 'concretions', which occur in the distal cells of the organ. It was also verified in the nuclei and in some cytoplasmic granules (lysosomes) of the proximal cells. The data indicated that lysosomes were involved with the nature or origin of the laminated concretions, but it is still questionable whether acid phosphatase activity exists in the nuclei.

Ultrastructural aspects of acid phosphatase activity in Malpighian tubules of Triatoma infestans Klug

Acid phosphatase activity was investigated ultrastructurally in Malpighian tubules of Triatoma infestans. Enzyme activity was demonstrated in laminated 'concretions' (distal cells) and in typical lysosomes, as well as in basal plasmalemma infoldings and basement membranes (especially in distal cells). This activity was assumed to be related to the excretory functions carried out mostly by the distal cells. Heterochromatin-nucleolus functional relationships involving RNA transcription may promote the nuclear reaction verified in the proximal cells and in some distal cells. A lead phosphate precipitate appeared free in the cytoplasm encircling the nuclei and was assumed to be a contamination from the nuclear precipitates.

KINETIC-PROPERTIES OF OSSEOUS PLATE ALKALINE-PHOSPHATASE FROM DIABETIC RATS

1. Increased levels of bone alkaline phosphatase activity were observed in diabetic rats. These animals exhibited impaired bone development without concomitant alterations of the sequence of cellular transformations.2. Alkaline phosphatase activity was delayed in diabetic rats but the kinetic parameters for the hydrolysis of p-Nitrophenylphosphate (PNPP) were virtually the same observed for controls (N = 1.2 and K0.5 = 43 muM).3. Alkaline phosphatase from diabetic rats had a better affinity (K0.5 = 38 muM) for magnesium ions than controls (K0.5 = 9 1 muM).4. Zinc ions affected alkaline phosphatase activity from control and diabetic rats in the same way (K0.5 = 10 muM).