nitrophenols and tartaric-acid

Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.

Topics: Amino Acid Sequence; Catalytic Domain; Crystallography, X-Ray; Humans; Malonates; Models, Molecular; Molecular Sequence Data; Nitrophenols; Organophosphorus Compounds; Phosphoric Monoester Hydrolases; Tartrates; Water

High-scale purification methods are required for several protein studies such as crystallography, mass spectrometry, circular dichroism, and function. Here we describe a purification method for PAP based on anion exchange, L-(+)-tartrate affinity, and gel filtration chromatographies. Acid phosphatase activity and protein concentration were measured for each purification step, and to collect the fractions with the highest acid phosphatase activity the p-nitrophenyl phosphate method was used. The purified protein obtained by the procedure described here was used for the determination of the first reported three-dimensional structure of prostatic acid phosphatase.

Topics: Acid Phosphatase; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Humans; Male; Nitrophenols; Organophosphorus Compounds; Prostate; Protein Tyrosine Phosphatases; Substrate Specificity; Tartrates

A recombinant putative acid phosphatase from Thermus thermophilus was expressed and purified from Escherichia coli. The recombinant phosphatase displayed activities in a broad range of temperature, from 40 to 90 degrees C, with optimal temperature at 70 degrees C. In addition, the recombinant enzyme had activities in a wide range of pH, from 3.6 to 9.1, with optimal pH at 6 in acetate buffer and with optimal pH at 6.5 in Hepes buffer. Furthermore, it showed significant thermal stability and still possessed 44% residual activity after 70 degrees C treatment for 15 min. Moreover, the recombinant phosphatase showed broad substrates specificities for monophosphate esters, p-nitrophenyl phosphate (pNPP) being the most preferred substrate, and it was able to resist inhibition by sodium tartrate. Additionally, the recombinant protein formed stable oligomer under partially denatured conditions and required calcium ions for enzymic activity.

Topics: Acid Phosphatase; Escherichia coli; Nitrophenols; Organophosphorus Compounds; Phosphoric Monoester Hydrolases; Recombinant Proteins; Substrate Specificity; Tartrates; Temperature; Thermus thermophilus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65 degrees C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a Km for p-nitrophenylphosphate and fructose-6-phosphate of 370 microM and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.

Topics: Acid Phosphatase; Adenosine Monophosphate; Agaricus; Chemical Fractionation; Chromatography, Gel; Chromatography, Ion Exchange; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fructosephosphates; Fungal Proteins; Glucose-6-Phosphate; Glycerophosphates; Glycoproteins; Isoelectric Point; Mannitol Phosphates; Molecular Weight; Nitrophenols; Organophosphorus Compounds; Phosphates; Polyethylene Glycols; Precipitin Tests; Protein Subunits; Substrate Specificity; Tartrates; Temperature

Recent evidence that Trypanosoma brucei synthesizes stage-regulated phosphotyrosine containing proteins and protein kinases stimulated us to assay bloodstream and insect stages of Trypanosoma cruzi and both pleomorphic and monomorphic clones of T. brucei for tyrosine phosphatase activity. Bloodstream and procyclic insect stages of T. brucei contained a 55-kDa protein that cross-reacted with monoclonal antibodies directed against the human placental tyrosine phosphatase PTP1B. Protein lysates of all life cycle stages of both trypanosomes dephosphorylated a nonspecific substrate, pNPP, and the specific substrate Tyr(P)Raytide. Dephosphorylation of Tyr(P)Raytide was effectively inhibited only by sodium vanadate, a specific phosphotyrosine phosphatase inhibitor, but pNPP activity was also inhibited by sodium fluoride (NaF) in lysates of T. brucei and by NaF and sodium tartrate in lysates of T. cruzi, suggesting that their respective lysates also contained serine/threonine and acid phosphatase activities. Fractionation studies revealed that most of this activity was in the cytosol. Stage regulation of tyrosine phosphatase activity in T. cruzi was strongly suggested by differences in the optimal pH for tyrosine phosphatase activity (7.0 for amastigotes and epimastigotes; 5.0 for trypomastigotes). We conclude that both species of trypanosomes synthesize tyrosine phosphatases and propose that identification and characterization of the enzymes responsible for this phosphatase activity could provide information about trypanosomal virulence or the regulation of trypanosomal growth and differentiation.

Topics: Animals; Antibodies, Monoclonal; Cytosol; Enzyme Inhibitors; Hydrogen-Ion Concentration; Immunoblotting; Indicators and Reagents; Nitrophenols; Organophosphorus Compounds; Peptides; Phosphorylation; Protein Tyrosine Phosphatases; Sodium Fluoride; Substrate Specificity; Tartrates; Trypanosoma brucei brucei; Trypanosoma cruzi; Vanadates

Acid phosphatases were examined histochemically at the light- and electron-microscopic levels using para-nitrophenyl phosphate (pNPP) and beta-glycerophosphate (beta-GP) as substrates. By light microscopy, there was intense activity with pNPP in supranuclear and distal regions of the secretory ameloblast, and moderate or slight activity respectively in those regions with beta-GP. These enzyme activities were less at the late secretory stage of amelogenesis and disappeared at the transitional stage. By electron microscopy, acid phosphatase activity was seen in the trans side cisternae of the Golgi apparatus, in lysosome-like granules, and in small vesicles in the Tomes' processes. The activity with pNPP but not beta-GP was also localized at the plasma membrane (proximal, lateral and distal surface). Activity with beta-GP was completely inhibited by 1 mM sodium tartrate and by 1 mM NaF; activity with pNPP was inhibited by 1 mM NaF and 10 mM sodium tartrate, but not by 1 mM sodium tartrate. Thus there are at least two different acid phosphatases, one tartrate-sensitive and the other 1 mM tartrate-resistant, in the secretory ameloblast; the tartrate-resistant enzyme is plasma-membrane bound.

Topics: Acid Phosphatase; Ameloblasts; Amelogenesis; Animals; Cell Membrane; Cytoplasmic Granules; Glycerophosphates; Golgi Apparatus; Histocytochemistry; Microscopy, Electron, Scanning; Molar; Nitrophenols; Organophosphorus Compounds; Rats; Rats, Inbred Strains; Tartrates; Tooth Germ; Vacuoles