Root surface acid phosphatase activities of vascular epiphytes of a Costa Rican rain forest

The objective of this work was to compare root surface phosphatase activities of vascular epiphytes typical of a lowland tropical forest. Acid phophatase, measured at pH 5.0 with the substrate p-nitrophenyl phosphate, was detected in 22 species distributed within 10 plant families. Epiphytes were classified as trunk, canopy-mat or bare-limb species based upon their usual occurrence. Phosphatase activity was not significantly correlated with plant occurrence. However, phosphatase activity was generally highest in trunk occurring and canopy-mat epiphytes rooted in mosses and humus-like accumulations, and lowest in species restricted to bare limbs. Epiphyte shoot phosphorus and chlorophyll content were correlated with species occurrence, with phosphatase being positively correlated with plant P content. The observed changes in acid phosphatase production among habitats were consistent with predicted changes in the availability of organic P sources. However, observed changes also reflect accompanying shifts in root structure including: the occurrence of velamen or waxy layers, changes in root diameter, branching and root hair density.     

Phosphatase Activity of Anaerobic Organisms

Anaerobic organisms were tested for phosphatase activity in different pH ranges. Several groups of organisms displayed characteristic patterns. Bacteroides fragilis, B. melaninogenicus, and B. ruminicola produced phosphatase with strongest activity at pH 8.6. Fusobacterium mortiferum was the only species of this genus to show strong hydrolysis. The enzyme was active in both acid and alkaline ranges. The activity of gram-positive organisms was variable, the most active groups being Clostridium perfringens, Peptostreptococcus intermedius, P. micros, and Peptococcus constellatus. The incorporation of phosphatase activity into the identification scheme of anaerobes seems feasible. There was a correlation of hydrolysis with several important pathogens.     

Synthesis and secretion of phosphatases by endophytic isolates of Colletotrichum musae grown under conditions of nutritional starvation

Even though fungal phosphatases are widely used to study ambient-regulated gene expression, little is known about these enzymes in the agriculturally important genus Colletotrichum. We have therefore identified several phosphatase activities in endophytic isolates of Colletotrichum musae grown under conditions of nutritional sufficiency or starvation for sources of phosphorus (P), nitrogen (N), carbon (C), and sulphur (S). These enzyme forms could be distinguished by substrate specificity, optimum pH, activation and inhibition by some substances, response to nutritional starvation, and pattern of migration in native gel electrophoresis. At least four individual phosphatase activities were identified under the growth conditions employed. A pH 5.0 acid phosphatase and an Mg(2+)-dependent pH 7.5 phosphodiesterase were expressed under all growth conditions at constant rates. Under conditions of P-starvation, derepression of a major pH 6.0-acid phosphatase was observed in cell-free extracts and the culture medium. A synthesis of alkaline phosphatase activities followed a more distinct pattern. Under conditions of nutritional sufficiency of P- or N-starvation, only a single intracellular enzyme form (optimum pH 10) was observed, which was resolved as a single electrophoretic activity band. However, in media lacking C or S sources additional alkaline phosphatase forms were derepressed with a concomitant increase in the overall enzyme activity level measured at pH 10. To our knowledge, this report represents the most detailed study of phosphatases in Colletotrichum and the first partial characterization of the phosphatase system in an endophytic fungus.     

Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.     

Effect of Phosphorus Deficiency on Phosphatase Activity of Cell Walls from Roots of Subterranean Clover

Clover (Trifolium subterraneum L. cv. Mt. Barker) was grownin solution culture with adequate (+P) or no phosphate (–P).Cell walls were extracted from roots in such a way that theywere uncontaminated by other cellular materials. Phosphataseactivity was assayed using p-nitro-phenylphosphate (NPP). Phosphatasebound to cell walls had a pH optimum between 5.0 and 6.0, irrespectiveof the P supply to the plants. Activity of phosphatase boundto cell walls increased with electrolyte concentration of theassay medium at pH 6.5 but not at pH 5.5. This increase in activitywas probably due to a higher degree of ionization of the cellwall at pH 6.5 than at pH 5.5, and to effects of high ionicstrength in decreasing the mutual repulsion of negatively chargedNPP from negative charges on the cell walls. Cell wall-boundphosphatase did not exhibit Michaelis-Menten kinetics: the concentrationof NPP at which activity was half the maximum rate (S_0.5) was0.7 mM for cell walls extracted from roots of both +P and –Pplants. Up to 30% of the phosphatase activity bound to cellwalls could be removed using buffer solutions of high pH andhigh ionic strength which contained Triton X100. Both soluble and cell wall-bound phosphatase(s) of roots increasedin activity with P deficiency. The phosphatase activity of cellwalls increased 1.5 fold as the P concentration in the rootsfell from 0.4–0.2% dry weight. Experiments with sterileroots of clover showed that increases in cell wall-bound phosphataseactivity associated with P deficiency were not due to microbialcontamination. It is argued that phosphatase(s) in cell wallsof roots could make a substantial contribution to the P nutritionof clover in soils deficient in inorganic phosphate by hydrolysingorganic phosphate compounds in the soil. Key words: Phosphatase, Clover, Roots, Phosphorus deficiency, Cell walls     

The acid phosphatases of Thermoascus crustaceus, a thermophilic fungus

Thermoascus crustaceus, a filamentous, thermophilic ascomycete with pathogenic potential was cultured on Sabouraud's liquid medium at temperatures from 27 to 47 degrees C for periods up to 7 days. Growth rate and yield were optimal at 37 degrees C. Morphological changes were confined to the cell walls, the thickness being greatest at 47 degrees C, which were also more resistant to mechanical disruption. Significant amounts of acid phosphatase (EC 3.1.3.2) activity occurred in the spent media of all cultures but were greatest at 37 degrees C. The proportions of acid phosphatase activity which were operationally defined as soluble or bound were also documented; the optimum pH for acid phosphatase activity in all fractions was 5.0. Extracts were subjected to polyacrylamide gel electrophoresis under non-denaturing conditions and the gels were stained for acid phosphatase activity. This revealed four electrophoretically distinct acid phosphatases which had different susceptibilities to inhibition by fluoride, phosphate, or tartrate. Effects of growth temperature, or phosphate supplement in the culture medium, on the acid phosphatase isoenzyme pattern were judged to be minor. Cytochemistry at the electron microscope level indicated acid phosphatase activity on the surface, in the periplasmic space, and in the cytoplasm, but no trends with regard to growth conditions. A substantial temperature range can be tolerated by this species but it is concluded that neither the general shape of the cells nor the acid phosphatase isoenzyme pattern changes substantially; this contrasts with previously documented differences for this class of enzyme in dimorphic Sporotrix schenckii.     

Phosphatase activity in relation to phytoplankton composition and pH in Swedish lakes

SUMMARY. 1. Potential phosphatase activity and phytoplankton from several lakes of different character were compared in order to evaluate the importance of lake water pH and phytoplankton composition for the activity and pH optimum of lake water phosphatases.
2. In oligotrophic lakes, in which phytoplankton biomass was most often dominated by Ochromonadaceae spp., optimum phosphate activity was found at pH values <6. In eutrophic lakes, where species of Cyanophyceae and Bacillariophyceae dominated the phytoplankton biomass, optimum phosphatase activity was found at pH 7.5 or 8.5.
3. The pH optimum of phosphatase activity often differed from the corresponding lake water pH.
4. Experimental variation in phosphorus availability resulted in predictable changes in phosphatase activity. However, specific phosphatase activity, calculated per biomass of phytoplankton, was dependent on plankton species composition.     

A major phosphotyrosyl-protein phosphatase from bovine heart is associated with a low-molecular-weight acid phosphatase

The phosphotyrosyl [Tyr(P)]-immunoglobulin G (IgG) phosphatase activity in the extracts of bovine heart, bovine brain, human kidney, and rabbit liver can be separated by DEAE-cellulose at neutral pH into two fractions. The unbound fraction exhibits a higher activity at acidic than neutral pH while the reverse is true for the bound fraction. Of all tissues examined, the Tyr(P)-IgG phosphatase activity in the unbound fraction measured at pH 5.0 is higher than that in the bound fraction measured at pH 7.2. The acid Tyr(P)-IgG phosphatase activity has been extensively purified from bovine heart. It copurified with an acid phosphatase activity (p-nitrophenyl phosphate (PNPP) as a substrate) throughout the purification procedure. These two activities coelute from various ion-exchange and gel filtration chromatographies and comigrate on polyacrylamide gel electrophoresis, indicating that they reside on the same protein molecule. The phosphatase has a Mr = 15,000 by gel filtration and exhibits an optimum between pH 5.0 and 6.0 when either Tyr(P)-IgG-casein or PNPP is the substrate. It is highly specific for Tyr(P)-protein with little activities toward phosphoseryl [Ser(P)]- or phosphothreonyl [Thr(P)]-protein. The enzyme activities toward Tyr(P)-casein and PNPP are strongly inhibited by microM molybdate and vanadate but insensitive to inhibition by L(+)-tartrate, NaF, or Zn2+. The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.     

Root surface phosphatase activity in ecotypes of Aegilops peregrina

The relationships between root surface phosphatase activity and the edaphic factors of their native habitats were investigated in four ecotypes of Aegilops peregrina (Hack.) Maire et Weil. In one set of experiments plants were grown in phosphate-deficient nutrient solution cultures (5 μ M ) with three pH values: 5.5, 6.5 and 7.5. In a second series, plants were grown in both P-poor and P-rich soils.
Results showed an optimal activity of the commonly-described root surface acid phosphatase of pH 4.5–5.0 in the ecotypes Meron (a P-poor montmorillonitic, typical mediterranean Terra-Rossa soil) and Har-Hurshan (a P-rich calcareous soil). However, in the ecotypes Malkiya (a P-rich kaolinitic Terra-Rossa) and Bet-Guvrin (a P-rich calcareous soil) the optimal activity of the phosphatase occurred at pH 6.0. The pH level of the growth solution had no effect on the pH of optimal activity of the phosphatase in the ecotypes Malkiya and Bet-Guvrin, but it somewhat affected their level of activity.
Phosphatase activity was stimulated when plant roots were grown in a P-poor soil, as compared to the activity of those which were grown in a P-rich soil. Plants of the Malkiya ecotype exhibited the strongest activation of phosphatase as compared to the other three ecotypes. It seems that ecotypes which have evolved in P-rich soils may regulate their root surface phosphatase activity better than those which have evolved in P-poor soils.     

Histochemistry of normal and diseased human lymph nodes.

Gomori's metal precipitate technique was used to demonstrate the phosphatase activity of the human cervical lymph node in health and disease, using four different phosphate esters (sodium beta-glycerophosphate and adenosine triphosphate at pH 9, riboflavin 5'-phosphate at pH 9.2 and 5'-monophosphoric acid at pH 8.3). In fetal lymph nodes, using 5'-monophosphoric acid, an outstanding positive activity was noticed in the lymphatic follicles. With the other three substrates there was either no nodular reaction or just a narrow rim of positive activity around the follicles, the internodular tissue being negative with all four substrates used. With chronic non-specific lymphadenitis the enzyme hydrolysing the three substrates (beta-glycerophosphate, riboflavin 5'-phosphate and adenosine triphosphate) began to make their appearance. It seems that with lymphadenitis, a qualitative change of the phosphatase activity takes place. A special characteristic pattern of phosphatase activity has been described in both 'early' and 'caseating' tuberculous lymphadenitis. In malignant lymphomas it was noticed that no activity was encountered with any of the four substrates in reticulum cell sarcoma. However, in lymphosarcoma a positive activity was obtained when either beta-glycerophosphate or adenosine triphosphate substrates was used, to the extent that one can depend upon this characteristic phosphatase activity in differentiating between reticulum cell sarcoma and lymphosarcoma. However, no enzymatic activity was obtained when the other two phosphate esters were used.     

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.     

Comparative study on hydrolases in five species of Ochromonas (chrysomonadina).

Acid phosphatase and beta-glucosidase were shown to be present in five species of Ochromonas grown in organic media (O. danica, O. malhanesis, O. munuta, O. sociabilis and Ochromonas sp. 933/4). Acid phosphatase was found to have a pH optimum at 4.0 in O. danica, and at 5.1 in the four other species. No alkaline phosphatase was found in any of the above mentioned species. Beta-glucosidase in the species studied has a pH optimum at 4.6. Low alpha-glucosidase activity was found only in O. danica. Acid phosphatase in all the five species shows an increase in activity during the logarithmic phase of growth and a decrease during the early stationary phase. Beta-glucosidase shows a similar behavior only in O. danica.     

Phosphate solubilization by Penicillium spp. isolated from soil samples of Indian Himalayan region

A total of 246 fungal isolates representing 36 genera and 72 species were isolated from the soil samples collected from Indian Himalayan region. Twenty-one species belonged to the genus Penicillium alone. All the Penicillium species were screened for phosphate solubilizing activity on Pikovskaya agar at 21 °C. Eight species of Penicillium, exhibiting formation of halos (zone of solubilization) around the fungal colonies in qualitative plate assays, were selected for quantitative estimations. In quantitative estimations that were conducted upto day 30 (at 3 days interval), seven species of Penicillium brought maximum solubilization after day 15, while P. oxalicum showed maximum solubilization after day 21 of incubation. The increase in solubilization coincided with decrease in pH of the broth. Acid phosphatase activity was 1.5–2.0 times higher in comparison to alkaline phosphatase. Many of these species showed wide range of tolerance for temperature, pH and salt concentration.     

Mechanism of action of Mg2+ and Zn2+ on rat placental alkaline phosphatase. I. Studies on the soluble Zn2+ and Mg2+ alkaline phosphatases.

Rat placental alkaline phosphatase (EC 3.1.3.1), a dimer of 135,000 daltons, is strongly activated by Mg2+. However, Zn2+ has to be present on the apoenzyme to obtain this activation. Mg2+ alone is unable to reconstitute functional active sites. Excess Zn2+ which competes for the Mg2+ site leads to a phosphatase with little catalytic activity at alkaline pH but with normal active sites at acidic pH as shown by covalent incorporation of ortho-[32P]phosphate. Two enzyme species with identical functional active sites have been reconstituted that only differ by the presence of Zn2+ or Mg2+ at the effector site. A mechanism is presented by which alkaline phosphatase activity of rat placenta would be controlled by a molecular process involving the interaction of Mg2+ and Zn2+ with the dimeric enzyme molecule.     

岷江上游本地种油松和外来种辐射松造林对土壤磷的影响

通过测定岷江上游16年生本地种油松和外来种辐射松人工林下不同土壤层次中各形态磷素含量以及磷酸酶活力,阐述两种林分对土壤磷素含量及其分布的影响.结果表明,在各个土层中,两种林分下的土壤含水量、pH值、有机碳的含量以及微生物生物量磷均无显著差异.在0～20cm土层中,油松林与辐射松林土壤全磷、Al-P、Fe-P、Ca-P含量以及磷酸酶活力均无显著差异,而油松林土壤有效磷和有机磷显著高于辐射松林;在20～40cm土壤中,油松林土壤全磷、有效磷、有机磷、Al-P、Fe-P含量与辐射松林差异不显著,油松林土壤Ca-P含量、酸性磷酸酶和中性磷酸酶活力显著高于辐射松林;在40～60cm土壤中,油松林土壤除中性磷酸酶活力与辐射松林的差异不显著外,其余各形态磷素含量和酸性磷酸酶活力变化与20～40cm土壤中的一致.此外,随土壤深度的增加,两种人工林土壤各形态磷素含量、磷酸酶活力都呈降低的趋势.单从土壤磷的状况看,油松林土壤中磷素含量高于辐射松林.     

EXTRACTIVE AND ENZYMATIC ANALYSES FOR LIMITING OR SURPLUS PHOSPHORUS IN ALGAE

An extractive Procedure for detection of surplusstored phosphorus (luxury consumption) in algae and an enzymatic analysis for conditions of P-limited growth in algae have been evaluated. A simple 60-min boiling water extraction of algae known to contain surplus P separates essential P compounds and surplus-stored P compounds. Surplus P compounds can be measured in the extract as orthophosphate. Extracts of algae limited in their growth by the amount of available P contain little or no orthophosphate. Limitation of algal growth by P supply induces the enzyme alkaline phosphatase. The activity of this enzyme can be measured at pH 9 using p-nitro-phenylphosphate as substrate. Algae which were P-limited and contained no extractable orthophosphate have as much as 25 times more alkaline phosphatase activity than algae with surplus available P .     

Fungal extracellular phosphatases: their role in P cycling under different pH and P sources availability

Aims

The aim of this work is to analyse the effect of pH, fungal identity and P chemical nature on microbial development and phosphatase release, discussing solubilization and mineralization processes in P cycling.

Methods and Results

P solubilizing fungi (Talaromyces flavus, T. helicus L, T. helicus N, T. diversus and Penicillium purpurogenum) were grown under three pH conditions (6, 6·5 and 8·5) and with different inorganic (calcium, iron, aluminium and rock) and organic (lecithin and phytate) P sources. P solubilization, mineralization, growth and phosphatase production were recorded. Acid and neutral environments maximized fungal development and P recycling. P chemical nature changed the phosphatases release pattern depending on the fungal identity. Acid phosphatase activity was higher than alkaline phosphatases, regardless of pH or sample times. Alkaline phosphatases were affected by a combination of those factors.

Conclusions

P chemical nature and pH modify fungal growth, P mineralization and solubilization processes. The underlying fungal identity‐dependent metabolism governs the capacity and efficiency of P solubilization and mineralization. P solubilization and mineralization processes are interrelated and simultaneously present in soil fungi.

Significance and Impact of the study

This study constitutes a reference work to improve the selection of fungal bioinoculants in different environmental conditions, highlighting their role in P cycling.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Summary Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl phosphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at nearneutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membrances. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.Supported by the Medical Research Council of Great Britain     

EXTRACTIVE AND ENZYMATIC ANALYSES FOR LIMITING OR SURPLUS PHOSPHORUS IN ALGAE

An extractive procedure for detection of surplus-stored phosphorus (luxury consumption) in algae and an enzymatic analysis for conditions of P-limited growth in algae have been evaluated. A simple 60-min boiling water extraction of algae known to contain surplus P separates essential P compounds and surplus-stored P compounds. Surplus P compounds can be measured in the extract as orthophosphate. Extracts of algae limited in their growth by the amount of available P contain little or no orthophosphate. Limitation of algal growth by P supply induces the enzyme alkaline phosphatase. The activity of this enzyme can be measured at pH 9 using p-nitro-phenylphosphate as substrate. Algae which were P-limited and contained no extractable orthophosphate have as much as 25 times more alkaline phosphatase activity than algae with surplus available P.     

Identification of the phosphomonoesterases that hydrolyze lysopolyphosphoinositides in rat brain and liver

The phosphatase activities responsible for the sequential dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate (lysoPtdIns(4,5)P2) to lysophosphatidylinositol that precedes reacylation in rat brain and liver microsomes were characterized. LysoPtdIns(4,5)P2 and the intermediate lysophosphatidylinositol 4-phosphate (lysoPtdIns4P) were hydrolyzed by two distinct phosphatase activities which were distinguishable by their substrate and cation requirements. The lysoPtdIns(4,5)P2 phosphatase activity was Mg2+ dependent and partially inhibited by Ca2+, excess Mg2+, and cationic detergent (cetyltrimethylammonium bromide). Activity was maximal at neutral (brain) or slightly alkaline (liver) pH when the Mg2+/lysoPtdIns(4,5)P2 molar ratio was 1.0 in the presence of bovine serum albumin (1 mg.mL-1). LysoPtdIns4P phosphatase activity did not require divalent cations (not inhibited by EDTA). This activity was inhibited by Ca2+, Mg2+, and substrate concentrations above 0.2 mM. Maximum activity was observed over a broad pH range (6.0-8.5). Both activities were inhibited by lysophosphatidylinositol and lysophosphatidylcholine, but not other lysophospholipids. The lysopolyphosphoinositides are most likely hydrolyzed by the same phosphatases that act on the diacylpolyphosphoinositides, since PtdIns(4,5)P2 and PtdIns4P were also hydrolysed by Mg2+-dependent and cation-independent phosphatases, respectively. Activities with the diacylpolyphosphoinositides differed only in their requirement of detergents for maximum activity in vitro. Specific activities for the diacyl and "lyso" forms of each substrate were very similar when suitably optimized reaction mixtures were used. The subcellular distributions of the two phosphatase activities in both brain and liver were the same when acting on diacyl- or lyso-polyphosphoinositides, as was their response to inhibitors. Alkaline, acid, phosphoprotein, and inositol-1-phosphate phosphatases did not contribute substantially to the hydrolysis of either lysoPtdIns4P or lysoPtdIns(4,5)P2, since the activities were not significantly inhibited by cysteine, dithiothreitol, NaF, or LiCl.(ABSTRACT TRUNCATED AT 250 WORDS)