Photosynthetic production and exoenzymatic degradation of organic matter in the euphotic zone of a eutrophic lake

Photosynthetic production of organic matter, and its exoenzymaticdecomposition were studied in the euphotic zone of a naturallyeutrophic lake during early spring phytoplankton bloom, andafter its breakdown. Phytoplankton were the major biomass producerswhen algae were actively growing, and the algal fraction (>3.0µm) contributed on average 75–80% to the total biomassof microplankton. When the phytoplankton bloom began to declinebacterial biomass increased rapidly and, at the end of the bloom,bacteria contributed 48.7–69.98% to the total biomassof microplankton. The high bacterial abundance during phytoplanktonbloom breakdown followed the highest rates of glucose uptake,and the highest rates of alkaline phosphatase, leucine-amino-peptidase,ß-galactosidase and ß-glucosidase activities.The majority of enzyme activity was associated with the bacterialsize fraction of seston. The activities of free (dissolved inwater) exoenzymes were negligible. The synthesis of bacterialexoenzymes was under control of an induction/derepression mechanism,and depended on the amount of easily assimilable substrates,and/or the presence of polymeric organic compounds in the water,which served as substrates for exoenzymatic hydrolysis. Thetight metabolic coupling between bacterial exoenzymatic hydrolysisand uptake of low molecular weight substrates, and its ecologicalsignificance is discussed.     

The relationship of alkaline phosphatase, CaATPase, and phytase

Alkaline phosphatase, highly purified from bovine intestinal mucosa, has significant hydrolytic activity against phytate and CaATP. Phytase and CaATPase activities require quite different assay conditions than those which are optimal for conventional alkaline phosphatase substrates such as 4-nitrophenyl phosphate. We have used affinity chromatography and antibody recognition to demonstrate that the phytase and CaATPase activities are not due to contaminating enzymes, but are intrinsic activities of intestinal alkaline phosphatase. All of the phytase and CaATPase activities present in crude extracts of bovine intestinal mucosa can be accounted for by alkaline phosphatase. Apparently neither phytase nor CaATPase exist in this tissue as independent enzymes. Specific substrates which require assay conditions quite different from the conventional 4-nitrophenyl phosphate substrate may account for the physiological function of "alkaline phosphatase."     

Impact de l'échantillonnage sur la mésure des nucléotides adényliques (ATP,ADP, AMP) du microplancton

The influence of sampling and sample treatment upon adenylic nucleotide (ATP, ADP, AMP) content of microplankton is studied. Changes in light conditions during nigh-sampling and extracting do not induce significant variations, in the adenylic nucleotide content of microplankton or in energy charge values.The contribution of zooplankton (size up to 200 µm) to microplankton adenosine values can be neglected for inshore surface waters and traditional sample volumes (about one liter). This result can been explained by the low density of zooplankton in such a small sample volume and by differences in efficiency of the extracting method used.

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Impact de l'échantillonnage sur la mésure des nucléotides adényliques (ATP, ADP, AMP) du microplancton

     

Origin and production of phosphatases in the acid Lake Gårdsjön

The activity of acid phosphatases was followed for one year in Lake Gårdsjön as well as in the inlet and the outlet of the lake. A budget of the phosphatases was calculated, including an estimation of the production of phosphatases. The phosphatase activity was also measured in two basins upstream of L. Gårdsjön: the north basin and the south basin of L. Stora Hästevatten.The acid phosphatase activity was very high compared with reported alkaline phosphatase activities in other lakes. About 95% of the phosphatases in L. Gårdsjön was produced in the lake, and the production was highest in early summer.Small Chrysophyceae (< 10 µm) probably produced the majority of the acid phosphatases in the investigated lakes, and accordingly could be favoured in environments with low phosphorus supply due to their ability to produce large amounts of phosphatases.     

A simple, specific radiometric assay for 5'-nucleotidase.

A radiometric assay for 5′-nucleotidese (EC 3.1.3.5) has been developed, which is applicable for all 5′-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were found to be the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 m Tris-HCl, pH 7.4; subsequent elution of the 5′-nucleotide was then accomplished with 0.2 m sodium phosphate, pH 7.4. Differential measurement of 5′-nucleotidase can be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavalin A into the reaction mixture. It completely inhibits 5′-nucleotidase without effecting the phosphatases. The applicability of this assay has been demonstrated by studying the properties of 5′-nucleotidase present in a purified plasma membrane preparation from a rat tumor which is enriched with both 5′-nucleotidase and alkaline phosphatase.     

Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

Influence of phytoplankton fractions on growth and reproduction of tropical cladocerans

The influence of two seston fractions, < 20 µm (nanoplankton) and 20 µm (microplankton), on growth and reproduction of cladoceran species with different sizes, from Lake Monte Alegre, was evaluated through individual growth and life table experiments. Size, shape and other features of the algae in the fractions were described. The procedure of filtering lake water through a 20 µm net for seston fractionation caused mutual contamination. The smallest cladoceran species, Ceriodaphnia cornuta Sars and Moina micrura Kurz, produced larger clutch sizes and exhibited higher intrinsic rates of population growth (r) in the nanoplankton, despite contamination of their food by inedible algae. The largest species, Simocephalus mixtus Sars, produced larger clutch sizes in the microplankton. There were no differences in juvenile biomass growth between treatments for M. micrura and Daphnia gessneri Herbst, but lower value of the exponential growth rate (g) in the microplankton was significant for M. micrura. Fecundity (eggs/total female) of M. micrura was significantly lower in the microplankton, while D. gessneri did not reproduce in this fraction, at the end of growth experiments. Spines, colonies, cenobium, filaments, hard cell wall, and gelatinous sheaths, represented constraints to cladoceran reproductive performance, besides algae size. Microplankton contamination by nanoplanktonic algae, in the experiments, probably minimized the negative effect of inedible algae. Nanoplankton was more suitable for the smallest species and microplankton for the largest one.     

Appearance of different phosphatase forms and phosphorus partitioning in nodules of chickpea (Cicer arietinum L.) during development

Acid and alkaline phosphatase and phytase activities were determined in the bacteroid free fractions of chickpea (Cicer arietinum L.) nodules at 15 days intervals, from 40 days after sowing (DAS) to 85 DAS. In general, the activities and specific activity of both the acid and alkaline phosphatases declined at 55 DAS. Out of the various substrates studied, ATP was the best substrate for both phosphatases. Activities of phosphatases with glucose-6-phosphate and fructose-6-phosphate were low in comparison to these with fructose 1,6 bisphosphate. The efficiency of acid phosphatase for utilizing fructose 1,6 bis phosphate as a substrate increased with nodule development. A fructose 1,6 bis phosphate specific acid phosphatase with elution volume to void volume (Ve/Vo) ratio of around 2.0 was observed in mature nodules (80 DAS). Acid phosphatase at 40 DAS was resolved into two peaks which were eluted at Ve/Vo of about 1.5 and 1.8. However, at 60 DAS the peak with Ve/Vo of 1.5 could not be detected. With ATP as substrate, a high (Ve/Vo of 1.2) and low MM form (Ve/Vo of 2.1) alkaline phosphatases were observed at 40 DAS however at 60 DAS stage only one peak with Ve/Vo of 1.7 was detected. Although, a low activity of acid phytase was observed in nodules at all stages of development but neither alkaline phytase nor phytic acid could be detected. It appears that the nodules acquire inorganic phosphate from the roots. The higher content of water soluble organic phosphorus in mature nodules could be due to the low activities of phosphatases at maturity.     

In situ bioassessment of dredging and disposal activities in a contaminated ecosystem: Toronto Harbour

The contamination of Toronto Harbour is a very serious problem. The major sources of pollution are the Don River and sewer outflows, as well as industrial, and municipal effluents. The problem is further compounded by perturbations of the toxic sediment caused by dredging, dredge-disposal, navigation, and recreational activities. The impact of contamination and nutrient enrichment was reflected in the size-fractionated primary productivity experiments. Generally, microplankton/netplankton (> 20 µm) productivity was enhanced whereas ultraplankton (< 20 µm) productivity was inhibited. These observations are attributable to interactions between ameliorating nutrients and toxic contaminants as well as to the differential sensitivity of natural phytoplankton size assemblages to the bioavailable chemical regime. In situ environmental techniques applied in Toronto Harbour were effective, sensitive, and rapid, and provided a better understanding of the impact of dredging/disposal activities under natural conditions. These techniques have great potential in the assessment of the ecotoxicology of harbours and other stressed environments.     

Further biochemical and morphological studies of granule fractions from rabbit heterophil leukocytes

Fractionation of rabbit heterophil leukocyte homogenates by isopycnic centrifugation as well as by zonal sedimentation has helped to characterize further the particulate components of these cells. Four classes have been identified: (A) Large (0.5–0.8 µm) and dense (1.26) azurophil or primary granules, containing all the myeloperoxidase, one-third of the lysozyme, and a major proportion of the lysosomal acid hydrolase activities of the cells. (B) Smaller (0.25–0.40 µm) and less dense (1.23) specific or secondary granules, containing 90% of the alkaline phosphatase and the remainder of the lysozyme activities, but very little if any acid hydrolases. (C) Particles of low density (1.20), containing the remainder of the lysosomal acid hydrolases. This fraction was heterogeneous, but showed abundant small rod- or dumbbell-shaped particles of moderate electron opacity, surrounded by a single membrane (tertiary granules?). The possible origin of these lysosomes from contaminating macrophages could not be ruled out but appeared unlikely. (D) Slowly sedimenting material of very low density (1.14), made up of large, empty vesicular membrane structures, and containing 10% of the alkaline phosphatase, and all of a thiol-dependent acid p-nitrophenyl phosphatase, an enzyme clearly different from the lysosomal acid phosphatase.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Alkaline phosphatase activity can interfere with the enzymatic determination of triglycerides

Unlike human plasma, rat plasma was found to contain substantial quantities of alkaline phosphatase. The large amount of phosphatase activity in rat plasma interferes with assay methods that employ orthophosphoric acid esters as substrates or reaction intermediates. Although these methods are effective when applied to human plasma samples, they cannot be used with rodent plasma.     

Phosphatase activity and phosphorus partitioning in nodules of developing mungbean

The acid and alkaline phosphatase activities were determined in bacteroid free fraction of nodules during development, using different phosphorylated substrates. Both enzymes change their substrate specificities with nodule development. Alkaline phosphatase, 20 days after sowing (DAS), showed negligible activity with ATP while at later stages maximum activity with ATP was observed. Invariably fructose 1,6 bisphosphate was a better substrate compared to fructose-6-phosphate and glucose-6-phosphate. Using Sephadex G-150 column chromatography, only one peak of acid phosphatase around Ve/Vo of 2.2 to 2.3 was observed at 20 and 30 DAS stages while at 40 DAS stage an additional ATP specific peak at around Ve/Vo of 2.9 was also observed. There was only one alkaline phosphatase peak at 20 and 30 DAS. However, at 40 DAS additional ATP specific peaks of phosphatases were observed at Ve/Vo of 1.4 and 2.6. Alkaline phosphatase could not be detected in the bacteroids whereas activity of acid phosphatase was about 5–7 % of that observed in the bacteroid free preparation. A low activity of both acid and alkaline phytases was observed at all stages of nodule development. However, phytic acid could not be detected. Increase in phosphorus content of water soluble organic phosphate at late stage of nodule development appears to be related with low level of phosphatase activity.     

The association of distinct acid phosphatases with the flagella pocket and surface membrane fractions obtained from bloodstream forms of Trypanosoma rhodesiense

Summary Cell fractionation of bloodstream Trypanosoma rhodesiense, using isopycnic sucrose gradient centrifugation, reveals acid phosphatase activities against a range of substrates to be associated, to varying degrees, with subcellular particle populations identified as derived from flagella pocket membrane and surface membrane. Using these same substrates ( and glycerophosphate, p-nitrophenyl phosphate and glucose-6-phosphate) at least two distinct acid phosphatase activities can be distinguished. One is thermolabile ( 80% inactivated after 30 min. at 60°C), sensitive to tartrate (50% inhibited at 1.8 mM Na tartrate) with a pH optimum 4.5 and appears to exhibit little substrate preference. The other acid phosphatase is relatively heat stable (30% inactivated), insensitive to tartrate (> 5.0% inhibited using 1.8 mM Na tartrate) exhibits a somewhat higher pH optimum ( 6.0) and is more substrate specific (6 × more active toward glucose-6-PO₄ than -glycerophosphate). Further cell fractionation experiments reveal 85% of the tartrate sensitive acid phosphatase to be associated with flagella pocket membrane and to account for 80% of the organisms hydrolytic activity toward -glycerophosphate. The tartrate resistant acid phosphatase however, has a much less exclusive localization being almost equally distributed between surface membrane (40%) and flagella pocket membrane (60%).     

Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid an alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrigfugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase an was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity, Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase an alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.     

Acid and alkaline phosphatases activities in vascular smooth muscle: species differences and subcellular distribution

1. Acid and alkaline phosphatase activities were studied in rat and dog aortic muscle using p-nitrophenyl phosphate (p-NPP) as the substrate. Alkaline phosphatase activity was quite comparable to acid phosphatase activity in rat aortic microsomes as well as further purified plasma membranes, but considerably lower than acid phosphatase activity in dog aortic membranes. 2. Subcellular distribution of acid and alkaline phosphatase activities in these vascular muscles indicated that alkaline phosphatases and a large portion of acid phosphatase activities were primarily associated with plasma membranes and the distribution of acid phosphatase showed little resemblance to that of N-acetyl-beta-glucosaminidase, a lysosomal marker enzyme. 3. The rat aortic plasmalemmal acid and alkaline phosphatase activities responded very differently to magnesium, fluoride, vanadate and EDTA. The alkaline phosphatase was more susceptible to heat inactivation than acid phosphatase. 4. These results suggest that these two phosphatases are likely to be two different enzymes in the smooth muscle plasma membranes. The implication of the present findings is discussed in relation to the alteration of these phosphatases in hypertensive vascular diseases.     

Seasonal changes in alkaline phosphatase activity of bacteria and microalgae in Lake Pavin (Massif Central,France)

Seasonal changes in alkalinephosphatase activity of bacteria and microalgae werestudied in the dimictic Lake Pavin (Massif Central,France), to test whetherthis activity is primarily algal or bacterial andwhether the APA presents seasonal variations coupledwith abiotic and biotic variables. Samples werecollected at different depths from May 1992 to May1993. The specific phosphatase activities wereanalysed spectrophotometrically with p-NitrophenylPhosphate (p-NPP) as substrate and were related to theprotein concentrations. No correlation was foundbetween alkaline phosphatase activity and solublereactive phosphorus (SRP) concentrations across anannual cycle. The specific activities of the smallclass (0.2–1.2 m) were the highest and thecontribution of this picoplanktonic size class(0.2–1.2 m) increased with depth. In addition, thelinear correlations between alkaline phosphataseactivity and protein concentration seemed to indicatethat most of these enzymes are constitutive. However,it cannot be excluded that the high phosphorusconcentrations repress APA.Finally, the measure of APA does not seem to be avalid quantitative test of the deficiency ofphosphorus for aquatic microorganisms.     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Depth profiles of plankton,particulate organic matter and microbial activity in the eastern Canadian Arctic during summer

Summary Deep profiles of particulate organic matter, microplankton (phytoplankton and bacteria), zooplankton and their metabolic activities were investigated during two summer voyages to the eastern Canadian Arctic. Magnitudes and depth distributions were similar in many respects to observations from temperate and tropical waters. Strong gradients in most properties were observed in the upper 50–100 m and subsurface maxima were generally associated with the upper mixed-layer (>50 m). In addition to the general vertical decreases in plankton biomass and metabolic activity there was evidence for both rapid transport (sinking) of organic matter and for enhanced (above background) levels of microbial metabolic activity in deep waters (>500 m). Zooplankton depth distributions differed from the pattern generally observed at lower latitudes; in the Arctic, zooplankton abundance decreased to a lesser degree with depth than particulate organics and microplankton. The overwintering behavior of high-latitude zooplankton appeared to be the best explanation for their relatively high abundance at depth. Despite this, however, zooplankton apparently contributed little to the total column community metabolism.