4-methylumbelliferyl Phosphate Research Articles

Isolation and characterization of Xenopus soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

A robust and quantitative method for tracking liposome contents after intravenous administration

We introduce a method for tracking the rate and extent of delivery of liposome contents in vivo based on encapsulation of 4-methylumbelliferyl phosphate (MU-P), a profluorophore of 4-methylumbelliferone (MU). MU-P is rapidly dephosphorylated by endogenous phosphatases in vivo to form MU after leakage from the liposome. The change in fluorescence spectra when MU-P is converted to MU allows for quantification of entrapped (MU-P) and released (MU) liposome contents by fluorescence or by a sensitive high performance liquid chromatography assay. We define the “cellular availability” of an agent encapsulated in a liposome as the ratio of the amount of released agent in the tissue to the total amount of agent in the tissue; this parameter quantifies the fraction of drug available for therapy. The advantage of this method over existing technologies is the ability to decouple the signals of entrapped and released liposome contents. We validate this method by tracking the circulation and tissue distribution of MU-P loaded liposomes after intravenous administration. We use this assay to compare the cellular availability of liposomes composed of engineered phosphocholine lipids with covalently attached cholesterol, sterol-modified lipids (SML), to liposomes composed of conventional phospholipids and cholesterol. The SML liposomes have similar pharmacokinetic and biodistribution patterns as conventional phospholipid–cholesterol liposomes but a slower rate of contents delivery into the tissue. Thus, MU-P enables the tracking of the rate and extent of liposome contents release in tissues and should facilitate a better understanding of the pharmacodynamics of liposome-encapsulated drugs in animals.

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.

Quality assurance in immunoassay performance—comparison of different enzyme immunoassays for the determination of caffeine in consumer products

Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.

4-Methylumbelliferyl phosphateを基質としたAlkaline phosphatase isoenzymeの検出法

4-Methylumbelliferyl phosphateを基質としたAlkaline phosphatase isoenzymeの検出法

In-gel phosphatase assay using non-denaturing two-dimensional electrophoresis

We developed a method for detecting phosphatase activities in crude tissue extracts after separation of proteins by a novel non-denaturing two-dimensional electrophoresis. In the first dimension, protein samples were separated by a MicroRotofor, a liquid-phase isoelectric focusing, in the presence or absence of urea. In the second dimension, fractionated proteins by the MicroRotofor were resolved by a native polyacrylamide gel electrophoresis in the presence of 20 mM 2-mercaptoethanol. After electrophoresis, the polyacrylamide gel was directly immersed in a reaction mixture containing 4-methylumbelliferyl phosphate (MUP), a fluorogenic substrate, and phosphatase activities were detected as fluorescent bands. In this assay, a variety of phosphatase activities were clearly detected in gel when the tissue extracts were separated by the MicroRotofor in the presence of 1.5 M urea. Furthermore, after detecting phosphatase activities in polyacrylamide gel at neutral pH, its activities at acidic pH could be detected by immersing the gel in sodium citrate buffer (pH 3.0). Therefore, this method is a quite useful technique to analyze various phosphatases by sequential reactions with MUP under different conditions after sample separation by the two-dimensional electrophoresis.

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Development of a microplate reader compatible microfluidic chip for ELISA

We report a novel microfluidic device use for sandwich enzyme-linked immunoassay assay (ELISA). The related procedures including the introduction of reagents, dilution and distribution of samples, as well as immobilization of enzyme can be readily carried out on a poly (dimethylsiloxane) (PDMS) chip. Particularly, this microfluidic chip comprising of two distinct parallel units, and has an identical dimension as a conventional microtiter plate, which offers access to the directly quantitative detection by the microplate reader. Gradient-concentration reacting solutions at six different concentrations level generated by the microfluidic channel network are simultaneously transported to 24 reaction chambers to form enzymatic products. Alkaline phosphatase (ALP), 4-methylumbelliferyl phosphate (4-MUP) and KH(2)PO(4) are used as enzyme-substrate-inhibitor model, to demonstrate the utility of the developed microchip-based enzyme inhibitor assay. Various conditions such as the surface treatment of chip channels, fluids velocities, substrate concentration, and buffer pH are investigated. The present microfluidic device for ELISA holds several advantages, for instance frugal usage of samples and reagents, less of operating time, favorably integrated configuration, ease of manipulation, and could be explored to a variety of high throughput drug screening.

Laser-induced fluorescence integrated in a microfluidic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori

A portable immunosensor coupled to laser-induced fluorescence (LIF) detection system has been successfully developed. It was applied to the quantitative determination of specific IgG antibodies against Helicobacter pylori present in human serum samples. This system is a relevant alternative tool for the diagnostic of peptic ulcer and gastritis diseases produced by H. pylori, which are recognized risk factors for the development of gastric mucosa associated lymphoid tissue lymphoma and gastric adenocarcinoma. More than 50% of the population has been affected by H. pylori, one of the most widespread infections around the world.Our proposed system combines the LIF detection, which offers great sensibility with the specificity of the immunological reactions and the microfluidic technology. The device has a central channel (CC) with packed H. pylori antigen immobilized on 3-aminopropyl-modified controlled pore glass (AP-CPG). Antibodies in serum samples reacted immunologically with the immobilized antigen and then, they were determined using alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The 4-methylumbelliferyl phosphate (4-MUP), employed as enzymatic substrate, was converted to soluble fluorescent methylumbelliferone by AP, and this fluorescent product was finally quantified by LIF detection system. The calculated detection limits for LIF detection and the ELISA procedure were 0.17 and 2.1UmL−1, respectively, and the within- and between-assay coefficients of variation were below 5.1%.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

Current standard assays using artificial substrates overestimate phosphodiesterase activity

Phosphodiesterases (PDE) are important enzymes in environments with significant proportion of phosphodiesters. The breakdown of phosphodiesters is a two step process, initiated by PDE hydrolyzing an ester-P bond on the diester molecule, breaking it into an organic moiety and a phosphomonoester. In the second step, the phosphomonoester is further hydrolyzed by phosphomonoesterase (PME) into the second organic moiety and free phosphate, Pi. The methods to assess extracellular PDE activities commonly use the substrate analogues bis-(p-nitrophenyl) phosphate (bis-pNPP) or bis-(4-methylumbelliferyl) phosphate (bis-MUFP), measure the resulting concentration of p-nitrophenol (pNP) or 4-methylumbelliferon (MUF), and assume the contribution from phosphomonoester hydrolysis by PME to be insignificant. To verify this, we measured concentrations of MUF and Pi at the end of the 30- and 60-min incubation of wetland plant root segments with a phosphodiester substrate. We found that the hydrolysis was complete (MUF:Pi ratio ∼ 2) confirming the importance of step 2. We suggest that a correction for additional hydrolysis by PME should be employed in samples where this is possible, such as plant roots or microbial cultures. Here, the MUF:Pi should be verified for each new sample type tested for its PDE activity, and values obtained from the PDE assay should be lowered accordingly. We do not recommend using the standard artificial substrates for PDE activity measurements in more heterogeneous samples, for example soils, where these assays have low reliability and can produce erroneous results.

In‐Gel Phosphatase Assay Using Fluorogenic and Radioactive Substrates

To investigate the regulatory mechanisms of cellular signaling by protein phosphorylation, it is important to analyze protein phosphatases, as well as protein kinases expressed in cells and tissues. In this unit, two different types of in-gel phosphatase assays are described. The first is an in-gel phosphatase assay using fluorogenic substrates. Protein samples containing phosphatase activities are resolved by native polyacrylamide gel electrophoresis (native-PAGE) and phosphatase activities detected in situ using fluorogenic substrates, such as 4-methylumbelliferyl phosphate (MUP) or 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). The other assay is an in-gel phosphatase assay using (32)P-labeled substrates. In this method, protein samples are resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using polyacrylamide gels containing (32)P -labeled substrates, renatured in situ, and the dephosphorylating activities detected by autoradiography. Each method has advantages and disadvantages that are discussed in the commentary.

Azadirachtin Interacts with Retinoic Acid Receptors and Inhibits Retinoic Acid-mediated Biological Responses

Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates

This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

ALKALINE PHOSPHATASE IN FRESHWATER CLADOPHORA-EPIPHYTE ASSEMBLAGES: REGULATION IN RESPONSE TO PHOSPHORUS SUPPLY AND LOCALIZATION1

Extracellular alkaline phosphatase enzyme activity (APA) is important for algal phosphorus (P) acquisition in P-limited freshwater ecosystems and is often used as an indicator of P deficiency. APA allows access to organic P (monophosphate esters), but the regulation of APA in response to availability of both PO43− and organic P is poorly characterized. This study aimed to examine the regulation of APA in freshwater Cladophora-epiphyte assemblages in response to PO43− and a hydrolyzable organic P source, and for the first time to apply enzyme linked fluorescence (ELF) to localize APA within freshwater macroalgal-epiphyte assemblages. In response to elevated PO43− concentrations, a component of net APA was suppressed, but there was also a constitutive APA, which was maintained even after prolonged exposure to nearly 1,000 μM PO43− and saturation of internal P pools. When supplied with organic glycerol P as the sole P source, the algae maintained APA in excess of needs for supplying PO43− for uptake, resulting in PO43− release into the medium. Constitutive APA may be adaptive to growth under chronic P limitation in oligotrophic freshwater habitats. Excess APA and release of PO43− could benefit different algal and bacterial partners within assemblages. APA in both Cladophora sp. and epiphytic algae was localized with ELF only when ethanol fixation was omitted. In algal subsamples exposed to different P treatments, there was no correlation between bulk APA (using 4-methylumbelliferyl phosphate [MUP] substrate) and % cell labeling with ELF, suggesting that ELF labeling of APA was at best semiquantitative in the algal assemblages.

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca 2+/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS–polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus–nematode interaction in liquid medium and fungus–nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS–PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus–nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus–nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.

Water-soluble octahedral polyammonium nanocapsules: synthesis and encapsulation studies

The syntheses of two water-soluble octahedral polyammonium nanocapsules 5e and 5f are described. Nanocapsule 5e was prepared via the TFA-catalyzed condensation reaction of six MOM-protected 4-hydroxybutyl-footed tetraformyl cavitands 3c with 12 ethylene diamines, followed by reduction and acidic hydrolysis of the intermediate octahedral polyimino nanocapsule. Nanocapsule 5f was prepared from 5e via the oxidation of the 4-hydroxybutyl feet to 3-carboxypropyl feet. Both nanocapsules are soluble in water below pH 5. 5f is also water-soluble above pH 7. In acidic aqueous solution, nanocapsule 5e encapsulates small negatively charged, partially hydrophobic guests, such as p-toluenesulfonic acid, N-BOC-aspartic acid, or 4-methylumbelliferyl phosphate. The single site microscopic binding constants for the latter guests are 150±30, 510±50, 1550±150 M −1, respectively and were determined from 1H NMR titrations and DOSY experiments by assuming a 6:1 binding model with six identical, independent binding sites per nanocapsule (statistical binding). 5e doesn't bind small neutral hydrophobic molecules in aqueous solution. However, both nanocapsules bind to nucleotides, such as ATP, dAMP, dGMP or TTP. NMR experiments support that nucleotides are not encapsulated, but bind to the outside of the nanocapsules.

Characterization of a unique class C acid phosphatase from Clostridium perfringens.

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Alkaline phosphatase activity of marine bacteria studied with ELF 97 substrate: success and limits in the P-limited Mediterranean Sea

The fluorogenic substrate Enzyme-Labeled Fluorescence 97 (ELF-P) is hydrolyzed by the P-cleaving enzyme phosphatase, producing ELF 97 alcohol (ELFA), a fluorescent-insoluble product. This reaction is used for monitoring phosphatase activity at the single-cell level. Most frequently, ELF-P has been used to determine the P-limitation status of microphytoplankton, but rarely of heterotrophic bacteria. We incubated ELF-P on filters to monitor marine bacterial cultures and oligotrophic Mediterranean Sea samples. Results were compared to classical measurements of bulk alkaline phosphatase activity using the fluorogenic substrate 4-methylumbelliferyl phosphate (MUF-P). A high percentage of the cultured cells were labeled with ELFA (the ratio of ELFA spots to total DAPI counts in P-limited cultures ranged from 26 to 100%, depending on the strain). In contrast, this ratio never exceeded 0.01 % in Mediterranean samples, even when P was demonstrated to be a significant limiting factor. This protocol is useful for application on cruises and with cultures, but was not sufficiently sensitive to detect P-stressed bacterial cells in oligotrophic marine environments.