nitrophenols and beta-glycerophosphoric-acid

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when β-GP was the sole source of Pi and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

Topics: Acid Phosphatase; Alkaline Phosphatase; Catalysis; Cations, Divalent; Glycerophosphates; Hydrogen-Ion Concentration; Hydrolysis; Levamisole; Nitrophenols; Organophosphorus Compounds; Substrate Specificity; Trypanosoma rangeli

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

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Alkaline Phosphatase; Animals; Enzyme Inhibitors; Female; Glycerophosphates; In Vitro Techniques; Nitrophenols; Oocytes; Organophosphorus Compounds; Suramin; Xenopus

The acid phosphate activity (APA) associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzed p-nitrophenyl phosphate (PNPP), pyrophosphate (PPi), and beta-glycerophosphate (beta GP). Inhibition of PNPP hydrolysis at pH 4.0 was inhibited in a competitive manner by the following compounds (listed in order of decreasing affinity with their apparent inhibitor constants (Ki')): molybdate (0.031 mM); PPi (0.147 mM); NaF (0.150 mM); o-carboxyphenyl phosphate (0.261 mM); inorganic phosphate (0.770)); arsenate (3.45 mM); tartrate (22.1 mM); and beta GP (29.8 mM). Cu2+, formaldehyde, and arsenite at 10:1, 80:1, and 200:1 inhibitor to substrate ratios did not inhibit APA. The maximal rate of hydrolysis (Vmax) of each substrate was greater at pH 4.0 than 5.0. The apparent Michaelis constant (Km') for PNPP increased from 0.233 to 0.351 mM when the pH was raised from 4.0 to 5.0. The Km' for PPi decreased from 0.101 to 0.046 mM, while the Km' for beta GP changed from 2.04 to 2.22 mM under similar circumstances. APA and alkaline phosphatase activity increased as a function of temperature up to 45 degrees C.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Cell Membrane; Diphosphates; Glycerophosphates; Hydrogen-Ion Concentration; Hymenolepis; Kinetics; Microvilli; Nitrophenols; Organophosphorus Compounds; Temperature

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

Intact spermatozoa from goat cauda epididymis possess phosphoprotein phosphatase activity that causes dephosphorylation of externally added [32p]histones. The enzymic reaction was linear with time for at least 15 min and there was little uptake of [32p]histones by these cells. The activity of the enzyme of the whole spermatozoa was not due to contamination of the broken cells or epididymal plasma and leakage of the intracellular enzymic activity during incubation. The activity of the phosphoprotein phosphatase was strongly inhibited by the thiol reagent: p-chloromercuribenzenesulfonic acid, which is believed not to enter the cells. There was no appreciable loss of the enzymic activity from the cells when washed with EDTA (2.0 mM) or a hyperosmotic medium. These data are consistent with the view that the observed activity of the enzyme is located on the spermatozoal external surface. Studies with unlabelled p-nitrophenyl phosphate and beta-glycerophosphate indicate that the sperm ecto-enzyme is not a non-specific phosphatase.

Topics: Animals; Cell Membrane; Chloromercuribenzoates; Edetic Acid; Epididymis; Glycerophosphates; Goats; Histones; Kinetics; Male; Nitrophenols; Organophosphorus Compounds; p-Chloromercuribenzoic Acid; Phosphoprotein Phosphatases; Phosphorylation; Sperm Motility; Spermatozoa

A cytochemical method for 5'-nucleotidase localization in which cerium serves as the capture agent in order to enzymatically detect liberated inorganic phosphate has been developed. The method has been established in cell-free model systems and in guinea pig neutrophils where 5'-nucleotidase is restricted to the plasmalemma as an ectoenzyme. This cerium-based method gives better results for ultrastructural localization of 5'-nucleotidase than conventional lead-based methods.

Topics: 5'-Nucleotidase; Adenosine Monophosphate; Alkaline Phosphatase; Animals; Cerium; Glycerophosphates; Guinea Pigs; Histocytochemistry; Male; Models, Biological; Neutrophils; Nitrophenols; Nucleotidases; Organophosphorus Compounds

Topics: Acid Phosphatase; Animals; Bone and Bones; Enzyme Activation; Femur; Glycerophosphates; Nitrophenols; Organophosphorus Compounds; Potassium Chloride; Rats; Sucrose; Tibia