Vertical variations in kinetics of alkaline phosphatase and P species in sediments of a shallow Chinese eutrophic lake (Lake Donghu)

The vertical distribution of the variables relevant to P forms in sediments were studied in a shallow Chinese freshwater lake (Lake Donghu) in 1997, 1998, 1999 and 2000, to assess the contribution of enzyme to P availability in sediment cores. Sediment P was fractionationd into iron-bound P, calcium-bound P, acid soluble organic P (ASOP) and hot NaOH extractable residual organic P. The former two species made the largest contribution to the sediment P pool. All P species exhibited significantly higher concentrations in different depths at Station I, compared with those found at Station II, except for ASOP. Coupled with these lower ASOP concentrations, the V _max data of alkaline phosphatase, measured on the same samples, were significantly higher at station I. Taken together, ASOP were probably important in supplying the enzymatic substrate (Phosphatase Hydrolyzable Phosphorus, PHP) into interstitial water. Dissolved orthophosphate and PHP concentrations were highly heterogeneous , but peaked in subsurface, paralleled by higher V _max and lower K _m values of alkaline phosphatase, throughout the sediment core. Sediment in the eutrophic lake is not only enriched in available P (iron-bound P), or stores residual P, but also tends to release PHP, thereby inducing the production of alkaline phosphatase and releasing o-P into water column by enzymatic hydrolysis. The latter process may also occur in relatively deep sediment layers.     

Characterization of ascorbyl esters hydrolysis in fish

The hydrolysis of ascorbate mono-, tri- and polyphosphates by trout intestinal alkaline phosphatase was examined. K_m values were established as 1.19, 4.1 and 3.7 mM, respectively. The enzyme catalyzed ascorbate triphosphate hydrolysis with 60% efficiency of that for ascorbate monophosphate. With the K_m value of 1.19 mM for ascorbate monophosphate the trout enzyme exhibits similar affinity with this substrate as with p-nitrophenyl phosphate (1.00–1.67 mM). Two K_m values for micro- and millimolar ranges of ascorbate monophosphate concentrations ranges were calculated as: 27.9 μM and 1.19 mM, respectively. Specific intestinal alkaline phosphatase inhibitor L-phenylalanine (100 mM), inhibited reaction rate by 20% in 10 min. We conclude that alkaline phosphatase, which is in a great abundance in the trout intestine, serves as ascorbate esters hydrolase, thus releasing active ascorbic acid into circulation.     

Aluminum sulfate treatment: Short-term effect on complex phosphorus compounds in a eutrophic lake

Application of aluminum sulfate to the hypolimnion of West Twin Lake, a hardwater eutrophic lake in northeastern Ohio, did not efficiently remove complex phosphorus compounds from the water column. The majority of the complex phosphorus compounds remaining after alum treatment released P0₄ following brief treatment with alkaline phosphatase.     

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10–1.15 g·cm^?3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5′-nucleotidase, had an apparent K_m for 4-methylumbelliferyl phosphate of approx. 59 μM, and was inhibited by levamisole. The high-density (1.20–1.25 g·cm^?3) peak was resistant to levamisole-inhibition, had an apparent K_m of approx. 30 μM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.     

Dynamics of water-extractable phosphorus during the degradation of Microcystis aeruginosa by four bacteria species

We carried out a laboratory experiment for studying the dynamics of water-extractable phosphorus release during the decomposition of dead Microcystis aeruginosa driven by the four bacteria strains. Our results showed that water-extractable phosphorus had significant correlation to alkaline phosphatase but without obvious correlation to acid phosphatase, suggesting that alkaline phosphatase was the primary enzyme decomposing organic phosphorus. Also, water-extractable phosphorus exhibited an obvious correlation to pH, indicating that the alkaline environment contributed to the release of water-extractable phosphorus. The quantity of bacteria hardly correlated to the release of water-extractable phosphorus for all species. This could result from decrease of bacterial quantity after consumption of substrate in closed system. Obvious variation in water-extractable phosphorus was observed across the strains in two water treatments. Sporolactobacillus sp. and Bacillus macerans had the strongest ability for decomposing organic phosphorus among five strain treatments in the lake water. In the distilled water, Bacillus sp. had the strongest ability to degrade organic phosphorus. In both water treatments, mixed species exhibited intermediate ability for decomposing organic phosphorus. It was true that water quality strongly affected the release of phosphorus by regulating phosphatase activity.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF A CALCIUM‐DEPENDENT ALKALINE PHOSPHATASE FROM THE CYANOBACTERIUM ARTHROSPIRA PLATENSIS 1

In the present study, Triton X‐114 (TX‐114) is used to extract and partially purify alkaline phosphatase (ALP) from a membranous fraction of Arthrospira platensis Gomont containing cell wall, plasma membrane, thylakoids, and sheath. TX‐114 has a double effect: solubilizing cell components to liberate the enzyme and, after phase partitioning, removing chl and other pigments present in the crude extract. The recovery of the enzyme in the aqueous phase suggests the overall hydrophilic character of this enzyme. ALP was kinetically characterized at pH 11.0 using p‐nitrophenyl phosphate as substrate, giving a K_m value of 1.7 mM. Orthovanadate was seen to be a competitive inhibitor of ALP, with a K_i of 0.8 mM. The enzyme was almost completely inactivated in the presence of 70 μM EDTA, although the addition of Ca²⁺ reverted this inactivation; these results indicate that ALP from A. platensis is a calcium‐dependent metalloenzyme. When the effect of Ca²⁺ was investigated in detail, a value of 0.067 μM^?1 for the affinity constant was obtained. The enzyme was histochemically localized in the cytoplasm, cell wall, and sheath using the enzyme‐labeled fluorescent substrate (ELF) method. It is assumed that the same enzyme is either soluble in the cytoplasm and in some way “trapped” in the cell wall or in the sheath. ALP localization within the sheath and the subsequent release of phosphorus (P) may benefit the neighboring cells surrounding this layer.     

Salinity-sensitive alkaline phosphatase activity in gills of the blue crab,Callinectes sapidus Rathbun

A levamisole-sensitive (K_i = 0.72 mM) alkaline phosphatase (pH optimum 9.1) and a levamisole-insensitive alkaline phosphatase (pH optimum 7.1) are present in gills of the blue crab Callinectes sapidus. Both enzymes are distinct from ouabain-sensitive ATPase. Specific activity for either phosphatase is greatest in the acinar tissue, which lines the branchial vessels. Histochemical localization of the enzymes confirmed this distribution. Activity of levamisole-sensitive alkaline phosphatase is affected by acclimation salinity. V_max of the levamisole-sensitive alkaline phosphatase is greater in high-salinity crabs than in low-salinity crabs; apparent K_m is not significantly different. The levamisole-sensitive alkaline phosphatase associated with the acinar tissue lining the branchial vessels may modulate the osmoregulatory response in blue crabs.     

Kinetic changes in surface phosphatase activity of Synedra acus (Bacillariophyceae) in relation to pH variation

• 1 The kinetics of surface phosphatase activity (PA) in the diatom Synedra acus were studied at low substrate concentrations that are frequently encountered in freshwaters and pH variations typical of hardwaters, ranging from pH 7 to pH 9. Higher pH resulted in higher values for the half saturation constant (K_m) and the maximal velocity (V_max).
• 2 The pH optimum of PA was shown to be a linear function of the logarithm of substrate concentration.
• 3 Similar slopes of Michaelis-Menten curves at different pHs in the range of low substrate concentrations indicate that the species is well adapted to hardwaters. The rate of release of phosphate from enzymaticaliy hydrolysable phosphorus was calculated from Michaelis-Menten kinetics for lake-water samples dominated by S. acus. Algal surface phosphatases were responsible for the hydrolysis of 1.54–1.64 nm min^?1 although incubation was performed at a lake temperature of only 7.3°C.

     

Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Characterization of orthophosphate release from dissolved organic phosphorus by gel filtration and several hydrolytic enzymes

The molecular weight distribution of dissolved organic phosphorus (DOP) and the possible mechanisms of orthophosphate (Pi) release were examined by gel filtration and incubation with some hydrolytic enzymes. Sixty five percent of the DOP appeared to have apparent molecular weights between 300 to 10000 daltons. Less than 10% of the DOP estimated higher molecules greater than 10000 daltons. Alkaline phosphatase released Pi more easily from low molecular weight (< 1500 daltons) DOP than from high molecular weight fractions. While, addition of nucleases or phosphodiesterase alone did not appear Pi release from high molecular weight DOP compounds. Pi release from those DOP compounds increased markedly (more than 30%) when alkaline phosphatase was incubated with nucleases or phosphodiesterase. However, 60% of DOP did not release Pi when alkaline phosphatase was incubated with either enzymes.     

Physiological variables determined under laboratory conditions may explain the bloom of Aphanizomenon ovalisporum in Lake Kinneret

Response of Aphanizomenon ovalisporum to certain environmental parameters was studied to gain a better understanding of the conditions which may have stimulated its autumnal bloom in Lake Kinneret. Optimal temperature for A. ovalisporum growth was 26–30?°C, resulting in growth rates of 0.2–0.3?day^?1, similar to those observed in the lake. Maximal rate of CO₂ fixation (assimilation numbers of 6–8?μg?C?μg^?1?Chl?h^?1) was obtained at low irradiances (I _k of 40–100?μmol?photons?m^?2?s^?1), 200?μM Pi and low N:Pi ratios. Growth was strongly affected by phosphorus availability, reaching a maximum at Pi concentrations above 40?μM. The high demand for phosphorus was indicated by an increase in alkaline phosphatase activity. The relative abundance of Pi in the cells increased by 4-fold in Pi-rich compared with Pi-limited cultures. Uptake of Pi was faster in Pi-depleted compared with Pi-sufficient cells. Maximal photosynthetic rates and K_1/2(HCO₃ ^?) were 140–220?μmol?O₂?mg^?1?Chl?h^?1 and 10–24?μM, respectively. At pH 7.0 the K _1/2(CO₂) was 2.2 and fell to 0.04?μM at pH 9.0. These data indicated that A. ovalisporum is a HCO₃ ^? user, and can explain its high photosynthetic rates during the bloom, under high pH and low dissolved CO₂ conditions. Na⁺ concentrations of about 5?mM were essential for A. ovalisporum growth at high pH approaching values in the lake.     

HOT WATER EXTRACTABLE PHOSPHORUS—AN INDICATOR OF NUTRITIONAL STATUS OF PERIDINIUM CINCTUM (DINOPHYCEAE) FROM LAKE KINNERET (ISRAEL)?1

The intracellular levels of hot water extractable and total phosphorus were determined in the dinoflagellate Peridinium cinctum. f. westii (Lemm.) Lef. for natural samples from the bloom in Lake Kinneret and from laboratory cultures. Amounts of phosphorus (P) in the hot water fraction, relative to total cellular phosphorus, were similar in lake Peridinium and in cells grown in high ambient orthophosphate (P_i) media (3–6 mg P · l^?1). The absolute amounts of hot water extractable P in natural cell and those cultured at lower P_i concentrations (0.02–0.05 mg P · 1^?1) were similar, although average P_i in lake water were 4 μg · l^?1. Under most growth conditions the hot water extract contained approximately equal amounts of molybdate reactive phosphorus (MRP) and non-MRP. Short chain (6–9 units) polyphosphates (mol wt 630–950) probably constituted the bulk of the non-MRP pool, which was hydrolysable by alkaline phosphatase and may serve as a precursor for a more permanent P store. Intracellular P levels and distribution were not directly dependent on external P_i concentrations but may be determined by the N:P atomic ratio or overall external ionic milieu. Peridinium grown in low ambient P_i released significant amounts of non-MRP compounds. In Lake Kinneret, for at least most of the bloom period, Peridinium does not appear to be limited by P supply.     

Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta

Protein tyrosine phosphatase 1B (PTP1B) appears to be an attractive target for the development of new drugs for type 2 diabetes and obesity. In our preliminary test, a MeOH extract of the stem barks of Sorbus commixta Hedl. (Rosaceae) showed strong PTP1B inhibitory activity. Bioassay?guided fractionation of the MeOH extract resulted in the isolation of two lupane?type triterpenes, lupenone (1) and lupeol (2). Compounds 1 and 2 inhibited PTP1B with IC₅₀ values of 13.7 ± 2.1 and 5.6 ± 0.9 μM, respectively. Kinetic studies revealed that both the compounds 1 and 2 are non?competitive inhibitors of PTP1B that decrease V_max values with no effect on K_m values.     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

Extracellular phosphatase activity in sediments of the Breitenbach,a Central European mountain stream

Activity of extracellular phosphatases (phosphomonoesterases) was measured in sandy streambed sediments of the Breitenbach, a small unpolluted upland stream in Central Germany. Fluorigenic 4-methylumbelliferyl phosphate served as a model substrate. Experiments were conducted using sediment cores in a laboratory simulation of diffuse groundwater discharge through the stream bed, a natural process occurring in the Breitenbach as well as many other streams.Streambed sediments contained high levels of particulate phosphorus, but concentrations of dissolved phosphorus in the interstitial water were 3 to 4 orders of magnitude lower. These interstitial concentrations were similar to those in the stream and groundwater. Extracellular phosphatase activity was high in the streambed sediments. These enzymes probably contribute significantly to the flux of phosphorus in sediment by hydrolyzing phosphomonoesters, making free phosphate available to the sediment microorganisms.Factors influencing the kinetic parameters V _max (maximum activity) and apparent K _m (enzyme affinity) of phosphatase were discharge rates of water through the sediment, water quality (ground- or stream water), and substrate (phosphomonoesters) as well as dissolved ortho-phosphate concentrations. Enzymes are supposed to be effective at limiting substrate concentrations, where, in this study, changes in discharge rates had little influence on rates of hydrolysis. Higher V _max and lower K _m values were found during percolation of groundwater through the sediment cores, compared with stream water. This indicates that rates of hydrolysis were higher with groundwater, both at substrate limitation and at substrate saturation. This was probably a consequence of the lower levels of dissolved ortho-phosphate in the groundwater.     

SIZE-DEPENDENT PHOSPHORUS UPTAKE KINETICS AND CELL QUOTA IN PHYTOPLANKTON1

The allometric equation, y = aX^b, described the interspecific variation of phosphate uptake kinetics and cell quota with phytoplankton cell size and showed that smaller cells are superior in uptake rate to large. Species-specific measurements, made by track autoradiography in phosphorus deficient cultures of communities from a phosphorus-limited lake, revealed that eight different species did not differ significantly in the Michaelis-Menten half-saturation constant, K_m. However, both saturated uptake rates (V_max) and the initial slope of the uptake curve (V_max:K_m) decreased per unit biomass with increasing cell size. Biomass-specific cell phosphorus quotas also decreased with increasing cell volume, but less rapidly than did V_max or V_max: K_m. Comparable data from the literature showed that marine species were superior in phosphorus uptake to freshwater species of similar size, but allometric variation of kinetics appeared to exist within both groups. Together with a variable internal stores model of phosphorus-limited growth, the allometric relationships of uptake kinetics and quotas predicted competition to favor smaller cells, with a differential in growth rate diminishing as competitive intensity increased.     

Stratification of alkaline phosphatase in sediments of two urban lakes and its effect on phosphorus cycle

Phosphorus loadings in sediments play an important role in lake eutrophication and the progress of its recovery. The phosphorus release is controlled by physical, chemical and biological mechanisms. Alkaline phosphatase catalyzes remineralization of organic phosphorus and then it may be an important factor accelerating phosphorus cycling in sediments. In this paper, distributions, properties and function of alkaline phosphatase with depths in sediments of two urban lakes were discussed. Alkaline phosphatase activity (APA) in the sediments of Lake Yuehu decreased with the sediment depth. APA in sediments of Lake Yuehu was, mostly, inhibited by Phe and L-Cys; and inhibiting ability of Phe could be stronger than L-Cys. APA in deeper layer (20–30 cm) of sediments was more sensitive to the inhibitors than other layers, but range of variation in APA was most wide in the subsurface layer (10–20 cm). All the facts implied that alkaline phosphatase occurred in various forms (isoenzymes). APA in the sediments with different depths of Lake Donghu responded Phe differently. Reacted with Phe and incubated for 1 day, the amounts of SRP released by these sediments varied correspondingly. SRP on the overlying water in deeper layers (5–10 cm and 15–20 cm) of Site T1 was higher than that in surface layer (0–5 cm) of the same site, 1 day after incubation. Hence, the SRP release resulted, at least partially, from the hydrolysis of some liable organic phosphorus mediated by APA. Alkaline phosphatase in lake sediments plays an important role in the release of internal phosphorus loadings and eutrophication. A possible explanation for the sensitivity at deeper layers could be another active region of hydrolysis by alkaline phosphatase from organic phosphorus, which added a new dimension in phosphorus cycling mediated by some biochemical mechanisms.     

Inhibition studies on the involvement of flavoprotein reductases in menadione- and nitrofurantoin-stimulated oxyradical production by hepatic microsomes of flounder (Platichthys flesus)

Inhibitors of mammalian cytochrome P450 and P450 reductase were used to investigate the enzymes in flounder (Platichthys flesus) hepatic microsomes involved in the stimulation of NAD(P)H-dependent iron/EDTA-mediated 2-keto-4-methiolbutyric acid (KMBA) oxidation (hydroxyl radical production) by the redox cycling compounds menadione and nitrofurantoin. Inhibitors were first tested for their effects on flounder microsomal P450 and flavoprotein reductase activities. Ellipticine gave type II difference binding spectra (app. K_s 5.36 μM; ΔA max 0.16 nmol-1 P450) and markedly inhibited NADPH-cytochrome c reductase, NADPH-cytochrome P450 reductase, and monooxygenase (benzo[a]pyrene metabolism) activities. 3-aminopyridine adenine dinucleotide phosphate (AADP; competitive inhibitor of P450 reductase) inhibited NADPH-cytochrome c but not NADH-cytochrome c or NADH-ferricyanide reductase activities. Alkaline phosphatase (inhibitor of rabbit P450 reductase) stimulated NADPH-cytochrome c reductase activity seven fold but had less effect on NADH-reductase activities. AADP inhibited nitrofurantoin- and menadione-stimulated KMBA oxidation by 45 and 17%, respectively, indicating the involvement of P450 reductase at least in the former. In contrast, ellipticine had relatively little effect, possibly because, unlike cytochrome c, the smaller xenobiotic molecules can access the hydrophilic binding site of P450 reductase. Alkaline phosphatase stimulated NAD(P)H-dependent basal and xenobiotic-stimulated KMBA oxidation, showing general consistency with the results for reductase activities. Overall, the studies indicate both similarities (ellipticine, AADP) and differences (alkaline phosphatase) between the flounder and rat hepatic microsomal enzyme systems.     

东北丘陵区林地转型耕地对土壤编码碱性磷酸酶基因的细菌群落的影响

【目的】通过研究林地转型耕地对土壤编码碱性磷酸酶基因的细菌群落丰度、多样性和结构的影响,为丘陵区耕地长期施肥下农田土壤微生物多样性丧失的影响机制以及未来的退耕还林过程中土壤微生物多样性的提升和土地可持续利用研究提供一些基础数据和技术支撑。【方法】采用实时荧光定量PCR (real-time quantitative PCR,qPCR)和高通量测序技术解析土壤编码碱性磷酸酶基因的细菌群落的丰度、多样性和结构变化,并耦合土壤化学性质分析,明确土壤编码碱性磷酸酶基因的细菌群落丰度和多样性与土壤化学性质的关系以及关键的驱动因子。【结果】林地垦殖为农田后,长期施肥导致土壤酸化,pH从5.58降至4.72,而土壤速效磷则从2.49 mg/kg增至49.3 mg/kg。相应地,耕地土壤编码碱性磷酸酶基因的细菌群落的丰度和Shannon指数均显著低于林地。基于编码碱性磷酸酶的phoD基因(alkaline phosphatase-encoding gene)序列的物种分类表明,丘陵区土壤编码碱性磷酸酶基因的细菌群落的优势门为变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、浮霉菌门(Planctomycetes)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)和疣微菌门(Verrucomicrobia),其中林地土壤的蓝藻门的相对丰度显著高于耕地。耕地土壤的慢生根瘤菌属(Bradyrhizobium)和芽孢杆菌属(Bacillus)的相对丰度显著高于林地,而中慢生根瘤菌属(Mesorhizobium)、假单胞菌属(Pseudomonas)、Chlorogloea属、Gemmata属、Phormidesmis属和Pseudolabrys属的相对丰度显著低于林地。土壤编码碱性磷酸酶基因的细菌群落结构因林地转型耕地而发生显著改变。phoD基因丰度和Shannon指数与pH显著正相关,而与总磷、速效磷、硝态氮和铵态氮均显著负相关,其中土壤速效磷是这些影响因素中影响最强烈的,长期施用无机磷肥导致含碱性磷酸酶的土壤细菌群落对有机磷分解的能力退化。【结论】林地转型耕地加之长期施肥改变了土壤pH和速效磷,并在其他理化因子的协同驱动下,导致土壤编码碱性磷酸酶基因的细菌群落丰度、多样性和结构的显著变化。