Phosphatases ofAcinetobacter lwoffi. Localization and regulation of synthesis by orthophosphate

Several phosphomonoesterases and diesterases with various pH optima have been observed inAcinetobacter lwofi JW11. The osmotic shock fluids contained only those with an alkaline pH optimum. The synthesis of these phosphatases was regulated by external Pi concentrations. The shock fluids were fractionated by chromatography, yielding three fractions, two of which had hydrophobic properties. One of these contained an alkaline phosphatase that specifically required Ca²⁺ for activity. The diesterases required various divalent cations for their function. Mutants that lack phosphomonoesterase or both phosphomonoesterase and phosphodiesterase activities were isolated.     

Phosphatase systems in Paramphistomum explanatum Fischoeder, 1901.

Extracts of adult Paramphistomum explanatum have been shown to contain high concentration of acid phosphomonoesterase with maximum activity at pH 4.5. The enzyme has been characterized by an exhibition of an unexpected increase in the inhibitory action of a mercury at 1 mM concentration by EDTA. With a lower concentration of mercury (0.1 mM and below) EDTA gave partial protection against inhibition. Different concentrations of magnesium and cobalt activated the enzyme while fluoride, copper, arsenate, tartrate and p-mercuribenzoate brought about inhibition. EDTA, glycine, glutathione and sodium azide had no effect. There was an indication of the presence of alkaline phosphomonoesterase at pH 10.0. The Km for p-nitrophenyl phosphate hydrolysis was 0.45 mM at pH 4.5.     

The cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental inorganic phosphate

Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.     

Localisation of phosphomonoesterase activity in ectomycorrhizal fungi grown on different phosphorus sources

Phosphorus (P) is a major limiting nutrient for plants in boreal forest ecosystems where a substantial part of the total P is sequestered in organic compounds. Some ectomycorrhizal (ECM) fungi are known to produce phosphomonoesterases, enzymes that degrade organic P sources. Here, we test 16 ECM species for this enzymatic activity by growing them on media containing orthophosphate, phytic acid or apatite. A method with an overlay gel that determined both phosphomonoesterase activity and its spatial distribution was developed. The phosphomonoesterase activity was not significantly higher when growing on organic P; conversely some isolates only produced measurable enzyme activity when grown on apatite. Species-specific variations with respect to phosphomonoesterase activity as well as growth responses to different substrates were found. The production of phosphomonoesterases was found to be widespread in ECM fungi and the enzyme activity did not need induction by organic P. The enzyme activity was highest in the central parts of the mycelia, potentially reflecting breakdown and recycling of phospholipids from old hyphae or potentially higher mycelial density.     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at low pH values

In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed by using PAGE of supernatant of the ameba homogenate obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the gel turned out to be sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium vanadate, ammonium molybdate, EDTA, EGTA, O-phospho-L-tyrosine, DL-dithiothreitol, H₂O₂, 2-mercaptoethanol, and ions of heavy metals—Fe²⁺, Fe³⁺, and Cu²⁺. Based on results of inhibitory analysis, lysosomal location in ameba cells, and wide substrate specificity of these two forms, it was concluded that they belonged to non-specific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosylprotein phosphatase activities. Two ecto-phosphatases were revealed in culture medium, in which amebae were cultivated. One of them was inhibited by the same reagent as the ameba tartrate-sensitive AcP and seemed to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed; it was not affected by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belongs to the ecto-ATPases found in many protists; however, so far its ability to hydrolyze ATP has not yet been proven.     

The development of phosphomonesterases in the testes of prepuberal chicks.

1. The biochemical development and histochemical localisation of phosphomonoesterases in the testes of prepuberal chicks have been studied. 2. Maximum acid phosphatase activity was observed at 12 weeks with a decrease in enzyme activity after this age, whereas alkaline phosphatase activity fluctuated with age. 3. Acid phosphatase activity in chicks was similar to that of the cockerel in being tartarate-insensitive. 4. There was a low level of significant correlation between acid phosphatase activity and testes weight. 5. Both alkaline and acid phosphatase activities were observed in the basement membrane of the seminiferous tubules, and acid phosphatase activity also in the various spermatogenic elements. 6. The results suggest that acid phosphatase is more involved in spermatogenesis, and more widely distributed than alkaline phosphatase in testicular tissue during testicular development.     

Hepatic and renal phosphomonoesterases and adenosine triphosphatases in chronic hyperprolactinaemic rats.

Influence of hyperprolactinaemia, induced endogenously by anterior pituitary transplantation on rat hepatic and renal cortical and medullary phosphomonoesterases and adenosine triphosphatases (ATPases) has been investigated. Although prolactin has a stimulatory effect on phosphomonoesterases and ATPases, it exhibits a specific and temporal influence on each subtype of hepatic and renal ATPases and phosphomonoesterases at different durations of pituitary transplantation. The specific activities of alkaline phosphatase and Na(+)-K+ dependent ATPases are activated in all the regions of different durations of experimentation. However, acid phosphatases, Ca2+ and Mg2+ dependent ATPases exhibit a differential response to prolactin in renal cortex, medulla and liver. Direct influence of prolactin on hepatic and renal phosphomonoesterases and ATPases is thus suggested.     

Phosphatases and phosphodiesterases isolated from the red king crab (Paralithodes camtschatica) hepatopancreas

Five enzymes have been isolated from the hepatopancreas of the red king crab Paralithodes camtschatica by means of ion exchange and gel chromatography: two acid (AcP) and one alkaline (AlkP) phosphomonoesterases, one alkaline phosphodiesterase (AlkPI), and one acid phosphodiesterase (AcPD). The pH optimum values of these enzymes are: AlkPs and AlkPD, 7.5; AcP, 5.5; and AcPD, 5.0. The activity of AlkP and AlkPD demands Mg²⁺ ions. The molecular weights of the enzymes (kDa) are the following: AlkP, 80; AcPs, 80 and 82; AlkPD, 51; and AcPD, 57. The enzymes are relatively thermostable (ID 50 from 47 to 62°C). AlkP is inhibited by NaCl (IC 50 at 0.4 M). The AcP, AcPD, and AlkPD activities are tolerant of high ionic strength.     

Coprecipitation of active uridine kinase and phosphomonoesterase from rat kidney by Zn2+-ions. III. Enzymes relevant to cancer chemotherapy.

Partially purified enzyme fraction from rat kidney possessing high uridine kinase and phosphomonoesterase activity was insolubilized by means of zinc precipitation without substantial loss of the activity. While uridine kinase in a soluble and Zn-precipitated form was inhibited by low concentrations (0.5-1.0 mM) of Zn2+-ions, phosphomonoesterase was fully active. In contrast to the soluble fraction, the two enzymes in zinc-precipitated and lyophilized preparations were stable on heating at 100 degrees C. Metal complexed proteins catalyze the dephosphorylation of 5'-UMP, 6-AzaUMP as well as of 2'(3')-UMP or 2,4-dinitrophenyl phosphate indicating thus the presence of several phosphomonoesterases in the complex.     

Effect of control of pH on the excretion of acid phosphatase by Rhodotorula glutinis

Summary The excretion of an acid phosphatase by Rhodotorula glutinis is related to the pH of the medium. During growth, the phosphatase excretion into the medium at a constant pH of 4.5 was 5 times higher than that observed at variable pH. After cultivation at a constant pH of 4.5 or at variable pH, cells were incubated at various pH values between pH 2 and 7. During this second incubation acid phosphatase release occured at pH 4.5 to 6.5 only. There was no release at pH 3.0; but when resting cells incubated at this pH were placed in a buffer solution at pH 5.5 a high activity was released. Extensive washing did not eliminate residual intrinsic acid phosphatase activity. These two types of acid phosphatase were phosphomonoesterases with an identical specificity for different substrates.     

草甸棕壤水稻田磷酸酶活性及对施肥措施的响应

1　引　　言土壤有机磷是一种重要的土壤磷素资源 .我国大部分土壤中有机磷占土壤全磷的 2 0 %～ 50 % ,但在森林和草原植被下的土壤可占到 50 %～ 80 % [9].土壤磷酸酶活性直接影响到有机磷库的利用 ,即磷酸酶活性是衡量土壤肥力 ,尤其是土壤有效磷水平的一个重要参考指标[15 ].土壤磷酸酶(Phosphatases)是催化含磷有机酯和酐水解的一类酶的总称 ,其活性高低直接影响着土壤中有机磷的分解转化及其生物有效性 .其中 ,磷酸单酯酶 (酸性、中性、碱性磷酸酶 )活性一直是土壤磷酸酶研究的重点[18].由于土壤中有机磷化合物的复杂性 ,除了磷酸单…     

Direct observation of the methionine residues of cytochrome c by 13C nuclear magnetic resonance spectroscopy.

The two Cepsilon-methyl methionine groups in cytochrome c have been chemically enriched (45%) with 13C. Their 13C NMR signals have been monitored in both the oxidized and reduced states and under various solution conditions. Methionine residue 80 showed characteristic chemical shift positions for the reduced Fe(II) and cyano-Fe(III) forms. No signal for methionine 80 was observed in the oxidized Fe(III) form due to the paramagnetic effect of the iron atom to which it is bonded, but the position of the methionine 65 signal was shifted, indicating that it is sensitive to the change of oxidation state. Two well resolved signals were observed at pH 11 for the Fe(III) form but only one was resolved at pH 2, indicating that while methionine 80 is definitely displaced from the iron atom at alkaline pH, it may not be in acid conditions.     

Alkaline phosphatase of the calf intestine hydrolyzes phospholipids

Pure alkaline phosphatase of the calf intestine is able to hydrolyze phosphatidylinositol 4,5-diphosphate (TPI) to phosphatidylinositol and Pi and to dephosphorylate phosphatidic acid. This phosphomonoesterase activity shows a considerably high specific activity when an incubation medium at neutral pH containing 3 mM deoxycholate is used. The activity is inhibited by low concentrations of Ca2+. The enzyme has no detectable phosphodiesterase activity under the conditions tested.     

Pyrococcus abyssi alkaline phosphatase: the dimer is the active form

Alkaline phosphatases (APs), E.C. 3.1.3.1, are non-specific phosphomonoesterases optimally active under alkaline conditions. They are classically known to be homodimeric metalloenzymes. This quaternary structure has been considered necessary for activity, although the relationship between quaternary structure and activity is not well understood. Recombinant Pyrococcus abyssi AP was previously isolated and characterized, appearing to have two active quaternary structures on native polyacrylamide gel electrophoresis: a monomer and a homodimer. The purpose of the present work was to determine the actual quaternary structure of P. abyssi AP in solution, by isolating each of the two quaternary forms and establishing the parameters governing the assembly and dissociation of the dimer. pH appeared to be an important parameter: in acidic media, the monomer/dimer ratio shifted towards monomer. Buffer composition also affected the quaternary structure: at the same pH, in potassium phosphate buffer, the two quaternary structures were observed, whereas in tris(hydroxymethyl)aminomethane hydrochloride buffer, only the dimer was observed. Metals bound to the enzyme were found to be involved in the stability of the quaternary structure. Indeed, the P. abyssi AP obtained upon removal of the metals was monomeric. Reactivation of the latter was achieved with variable efficiency. From these experiments, no active monomer could be isolated, leading the conclusion that the active form of P. abyssi AP is the homodimer.     

The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7-7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has K(m)1.43x10(-4)m. 2. A factor from the ;pH5 supernatant' of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ;pH5 supernatant'. Maximum stimulation was observed with the P(1) fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ;pH5 supernatant'. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5'-nucleotidase, but different from that of alkaline phosphatase.     

Milk fat globule membranes. Inhibition by sucrose of the alkaline phosphomonoesterase.

Sucrose, a widely used agent in the preparation of membranes, inhibited the alkaline phosphomonoesterase of the milk fat globule membrane in both its membrane-bound and detergent-solubilized forms. The inhibition was kinetically competitive and reversible by dialysis. However, its mechanism was more complex than simple competition with substrate because: (a) sucrose induced the appearance of prolonged time-lags in the progress curves of the enzyme; (b) the extent of inhibition and of the time-lags depended on the age of the membrane preparation, the period of pre-exposure of the membranes to sucrose, and the temperature of pre-exposure. On the other hand the acid phosphomonoesterase and the phosphodiesterase activities also present in the membrane preparations were unaffected by the disaccharide.     

Purification and characterization of thermo-labile alkaline phosphatase from an Antarctic psychrotolerant Bacillus sp. P9

A psychrotolerant Bacillus sp. from Antarctica produced an alkaline phosphatase in the culture supernatant. The strain showed 98.4% 16s rDNA sequence identity with Bacillus sphaericus. The 76 kDa protein was purified 11.1-fold showing alkaline phosphomonoesterase activity. Enzyme was optimally produced at 25 °C and pH 7.0. This cold active alkaline phosphatase is heat labile and gets completely inactivated at 60 °C in 50 min and is active in broad pH range.     

Milk fat globule membranes. Inhibition by sucrose of the alkaline phosphomonoesterase

Sucrose, a widely used agent in the preparation of membranes, inhibited the alkaline phosphomonoesterase of the milk fat globule membrane in both its membrane-bound and detergent-solubilized forms. The inhibition was kinetically competitive and reversible by dialysis. However, its mechanism was more complex than simple competition with substrate because: (a) sucrose induced the appearance of prolonged time-lags in the progress curves of the enzyme; (b) the extent of inhibition and of the time-lags depended on the age of the membrane preparation, the period of pre-exposure of the membranes to sucrose, and the temperature of pre-exposure. On the other hand the acid phosphomonoesterase and the phosphodiesterase activities also present in the membrane preparations were unaffected by the disaccharide.     

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal

Abstract We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M _r = 51 000 and L-AP, M _r = 39 500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.     

重金属污染区土壤酶活性变化

从福建龙岩新罗区特钢厂污灌区农田采集土壤,测定土壤基本理化性质及脲酶、纤维素酶、碱性磷酸酶、多酚氧化酶、过氧化氢酶活性和Cu、Cd、Pb、Zn含量,探讨重金属污染和土壤性质对土壤酶活性的影响.结果表明： 4种全量或有效态重金属与土壤脲酶、纤维素酶、碱性磷酸酶和多酚氧化酶活性呈显著正相关,与过氧化氢酶活性呈显著或极显著负相关;土壤pH与碱性磷酸酶活性呈极显著正相关,粉粒含量与过氧化氢酶活性呈显著负相关.经通径分析,重金属污染刺激了脲酶、多酚氧化酶和纤维素酶活性,但对碱性磷酸酶活性的影响较小.有效态Cu、Cd、Pb、Zn对过氧化氢酶活性的直接影响并不大,但通过间接途径抑制了过氧化氢酶活性.土壤理化性质对5种土壤酶活性的影响较大,碱解氮直接抑制了脲酶活性;全磷直接刺激了碱性磷酸酶和过氧化氢酶活性,并通过有效磷刺激了纤维素酶活性;有效磷直接刺激了纤维素酶活性,直接抑制了碱性磷酸酶和过氧化氢酶活性;全钾直接抑制了碱性磷酸酶和多酚氧化酶活性;速效钾通过有效磷刺激了纤维素酶活性;土壤颗粒组成明显影响多酚氧化酶和过氧化氢酶活性.5种酶活性与土壤Cu、Cd、Pb、Zn含量之间的关系不明确,因此其活性不是指示土壤Cu、Cd、Pb、Zn污染的良好指标.