Addition Of Alkaline Phosphatase Research Articles

Near-infrared light-induced photoelectrochemical biosensor based on plasmon-enhanced upconversion nanocomposites for microRNA-155 detection with cascade amplifications

Herein, a novel near-infrared (NIR) light-driven photoelectrochemical (PEC) biosensor based on NaYF4:Yb3+, Er3+@Bi2MoO6@Bi (NYF@BMO@Bi) nanocomposites was elaborately developed to achieve highly sensitive detection of microRNA-155 (miRNA-155). To realize signal enhancement, the coupled plasmonic bismuth (Bi) nanoparticles were constructed as an energy relay to facilitate the transfer of energy from NaYF4:Yb3+, Er3+ to Bi2MoO6, ultimately enabling the efficient separation of electron-hole pairs of Bi2MoO6 under the irradiation of a 980 nm laser. For constructing biosensing system, the initial signal was firstly amplified after the addition of alkaline phosphatase (ALP) in conjunction with the biofunctionalized NYF@BMO@Bi nanocomposites, which could catalyze the conversion of ascorbic acid 2-phosphate into ascorbic acid, and then consumed the photoacoustic holes created on the surface of Bi2MoO6 for the enlarging photocurrent production. Upon addition of target miRNA-155, the cascade signal amplification process was triggered while the ALP-modified DNA sequence was replaced and then followed by the initiation of a simulated biocatalytic precipitation reaction to attenuate the photocurrent response. On account of the NIR-light-driven and cascade amplifications strategy, the as-constructed biosensor was successfully utilized for the accurate determination of miRNA-155 ranging from 1 fM to 0.1 μM with a detection limit of 0.32 fM. We believed that the proposed nanocomposites-based NIR-triggered PEC biosensor could provide a promising platform for effective monitoring other tumor biomarkers in clinical diagnostics.

Fluorescence Turn-on Detection of Alkaline Phosphatase Activity and Al3+ Using Vitamin B6 Cofactor Conjugated GSH Capped Mn-doped ZnS Quantum Dots.

The glutathione (GSH) functionalized Mn-doped ZnS quantum dots (GSH_Mn_ZnS QDs) was conjugated with pyridoxal 5'-phosphate (PLP). The -CHO group of vitamin B6 cofactor PLP interacted with the -NH2 group of GSH functionalized Mn_ZnS QDs. The conjugation of PLP quenched the fluorescence emission of GSH_Mn_ZnS QDs at 601nm. Addition of alkaline phosphatase (ALP) catalytically dephosphorylated the PLP into pyridoxal that restored the fluorescence emission of GSH_Mn_ZnS QDs. With a sensitivity of 0.035 U/L, the PLP conjugated GSH_Mn_ZnS QDs was applied to quantify ALP activity in human serum and plasma. Further, the developed nanoprobe PLP conjugated GSH_Mn_ZnS QDs was also applied to detect Al3+. The complexation-induced fluorescence enhancement was observed at 492nm upon the interaction of Al3+ with the PLP conjugated GSH_Mn_ZnS QDs. Without any interference from other tested metal ions, this nanoprobe can be employed to detect Al3+ down to 2.30µM.

Detection of Pyrophosphate and Alkaline Phosphatase Activity Based on PolyT Single Stranded DNA - Copper Nanoclusters

The determination of pyrophosphate and alkaline phosphatase activity plays a significant role in medical diagnosis. In this work, a label-free "ON-OFF-ON" fluorescence strategy is developed for the analysis of pyrophosphate and alkaline phosphatase activity. Using PolyT single strand DNA as templates to synthesize fluorescent copper nanoparticles, the coordination effect of pyrophosphoric acid on Cu2+ inhibited the generation of fluorescence. Afterwards, the addition of alkaline phosphatase into hydrolyze pyrophosphoric acid resulted in the release of Cu2+, whereby the fluorescence intensity could be recovered. Thereupon enhanced-sensitivity for alkaline phosphatase was obtained (0.1 mU/L), much better than previously reported methods. Meanwhile, it could be performed directly in homogeneous solution, which was very close to the actual activity level of alkaline phosphatase under physiological conditions. Likewise, satisfactory results were also obtained in specificity assessment, which demonstrated its potential application in clinical diagnosis. Notably, a new, sensitive, low-cost, short-time, and high-sensitivity platform for alkaline phosphatase detection was constructed, and the design of biosensor using DNA-templated Copper nanoclusters (CuNCs) was instructed in this study.

Highly sensitive detection for alkaline phosphatase using doped ZnS quantum dots with room temperature phosphorescence and its logic gate function

This paper presents a highly sensitive sensing system for alkaline phosphatase by room temperature phosphorescence of Mn doped ZnS quantum dots and pyrophosphate. The sensing system has intense room temperature phosphorescence emission in the absence of alkaline phosphatase. The phosphorescence is quenched gradually with the addition of alkaline phosphatase. The emission “on” without alkaline phosphatase may be attributed to the increased probability of charge transfer from one of surface traps to the dopant bands of another resulted from the shortened dot-to-dot distance by the strong chelation of pyrophosphate and Zn2+ ion and the hydrogen bonding between pyrophosphate and β-cyclodextrin. The addition of alkaline phosphatase causes pyrophosphate hydrolyzed to orthophosphate and the dot-to-dot distance of quantum dots back to the normal, and then the phosphorescence “off”. The factors affecting the sensing system performance were also optimized. Under the optimal experimental conditions, the linear range for alkaline phosphatase is determined as 0.2−10 U/L with a LOD at 0.045 U/L. The recovery of human serum was determined from 93.75%–103.03%, indicating a potential application in biomedical diagnosis. Furthermore, an RTP-based “INHIBIT” logic gate using the doped ZnS quantum dots was also presented.

The Cyclic AMP Signaling Pathway and Direct PKA Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent disease caused by mutations in the genes encoding polycystins 1 and 2, which form a complex where the effect systems lie in polycystin-2 (PC2). PC2 is a TRP-type, Ca2+-permeable non-selective cation channel that plays an important role in Ca2+ signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue Serine 829, in the carboxy terminus that seems to be a gating mechanism of channel activity. The fact that the cAMP/PKA pathway participates in the regulation of PC2 channel function is crucial for the understanding of renal physiology and the pathogenesis of ADPKD.

The cAMP Signaling Pathway and Direct Protein Kinase A Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function

Polycystin-2 (PC2) is a TRP-type, Ca(2+)-permeable non-selective cation channel that plays an important role in Ca(2+) signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue serine 829, in the carboxyl terminus.

D-amino acids modulate the cellular response of enzymatic-instructed supramolecular nanofibers of small peptides.

Peptides made of d-amino acids, as the enantiomer of corresponding l-peptides, are able to resist proteolysis. It is, however, unclear or much less explored whether or how d-amino acids affect the cellular response of supramolecular nanofibers formed by enzyme-triggered self-assembly of d-peptides. In this work, we choose a cell compatible molecule, Nap-l-Phe-l-Phe-l-pTyr (LLL-1P), and systematically replace the l-amino acids in this tripeptidic precursor or its hydrogelator by the corresponding d-amino acid(s). The replacement of even one d-amino acid in this tripeptidic precursor increases its proteolytic resistance. The results of static light scattering and TEM images show the formation of nanostructures upon the addition of alkaline phosphatase, even at concentrations below the minimum gelation concentration (mgc). All these isomers are able to form ordered nanostructures and exhibit different morphologies. According to the cell viability assay on these stereochemical isomers, cells exhibit drastically different responses to the enantiomeric precursors, but almost same responses to the enantiomeric hydrogelators. Furthermore, the different cellular responses of LLL-1P and DDD-1P largely originate from the ecto-phosphatases catalyzed self-assembly of DDD-1 on the surface of cells. Therefore, this report not only illustrates a new way for tailoring the properties of supramolecular assemblies, but also provides new insights to answering the fundamental question of how mammalian cells respond to enzymatic formation of nanoscale supramolecular assemblies (e.g., nanofibers) of d-peptides.

Tissue-nonspecific alkaline phosphatase promotes axonal growth of hippocampal neurons

Axonal growth is essential for establishing neuronal circuits during brain development and for regenerative processes in the adult brain. Unfortunately, the extracellular signals controlling axonal growth are poorly understood. Here we report that a reduction in extracellular ATP levels by tissue-nonspecific alkaline phosphatase (TNAP) is essential for the development of neuritic processes by cultured hippocampal neurons. Selective blockade of TNAP activity with levamisole or specific TNAP knockdown with short hairpin RNA interference inhibited the growth and branching of principal axons, whereas addition of alkaline phosphatase (ALP) promoted axonal growth. Neither activation nor inhibition of adenosine receptors affected the axonal growth, excluding the contribution of extracellular adenosine as a potential hydrolysis product of extracellular ATP to the TNAP-mediated effects. TNAP was colocalized at axonal growth cones with ionotropic ATP receptors (P2X₇ receptor), whose activation inhibited axonal growth. Additional analyses suggested a close functional interrelation of TNAP and P2X₇ receptors whereby TNAP prevents P2X₇ receptor activation by hydrolyzing ATP in the immediate environment of the receptor. Furthermore inhibition of P2X₇ receptor reduced TNAP expression, whereas addition of ALP enhanced P2X₇ receptor expression. Our results demonstrate that TNAP, regulating both ligand availability and protein expression of P2X₇ receptor, is essential for axonal development.

ChemInform Abstract: Synthesis of Water-Soluble Prodrugs of the Cytotoxic Agent Combretastatin A4.

Water-soluble phosphate and glycine carbamate prodrugs of the cytotoxic agent Combretastatin A4 (1) have been prepared. The phosphate prodrug was degraded slowly in plasma at 37°C. The degradation was accelerated by the addition of alkaline phosphatase.

Ectoprotein kinase-mediated phosphorylation of FAT/CD36 regulates palmitate uptake by human platelets.

Glycoprotein IV (FAT/CD36) has been shown to be phosphorylated by a cAMP-dependent, platelet membrane-bound ectokinase. In this study, we demonstrate that ectophosphorylation of FAT/CD36 regulates initial palmitate uptake. This is the first time that short-term regulation of the activity of a long-chain fatty acid carrier could be shown. Phosphorylation of FAT/CD36 was paralleled by a significant decrease in initial palmitate uptake by morphologically and functionally intact platelets. Maximum inhibition of palmitate uptake was achieved at 0.5 nM extracellular ATP, being significantly decreased to 72% compared to the control. Inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI or with beta,gamma-methylene-ATP, and was reversible upon addition of alkaline phosphatase. An extracellular ATP concentration above 5 microM completely prevented the ectophosphorylation-mediated inhibition of palmitate uptake. We conclude that FAT/CD36-mediated palmitate uptake by human platelets is short-term regulated via cAMP-dependent ectophosphorylation of FAT/CD36.

Hydrolyzable ATP and PIP(2) modulate the small-conductance K+ channel in apical membranes of rat cortical-collecting duct (CCD).

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATPγS and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.

INFLUENCE OF PHOSPHORYLATION ON PROTEOLYTIC CLEAVAGE

ABSTRACT Phosphorylation of an amino acid residue close to the peptide bond, which is a target of a specific proteolytic enzyme, seems to cause inhibition of such a cleavage. This finding may have a number of important consequences. The effect of phosphorylation of a model peptide on its proteolytic cleavage by trypsin was studied. The velocity of peptide bond cleavage of nonphosphorylated synthetic peptide was compared with cleavage velocity of the same bond of synthetic peptide phosphorylated on serine residue located in closed proximity of the specifically cleaved peptide bond. It was shown that the enzymatic cleavage was inhibited by phosphorylation of mentioned amino acid residue. Reversed-phase high-performance chromatography (RP-HPLC) was used for the fast and reliable analysis of reaction mixtures. Influence of the addition of alkaline phosphatase on proteolytical cleavage of casein was also investigated. RP-HPLC peptide maps of β-casein cleaved by trypsin in the presence and absence of alkaline phosphatase were compared. It was proven that the action of acid phosphatase affects the composition of the resulting proteolytic digest.

Synthesis of water-soluble prodrugs of the cytotoxic agent Combretastatin A4

Water-soluble phosphate and glycine carbamate prodrugs of the cytotoxic agent Combretastatin A4 ( 1) have been prepared. The phosphate prodrug was degraded slowly in plasma at 37°C. The degradation was accelerated by the addition of alkaline phosphatase.

Mg 2+-dependent phosphatase as an inhibitory mediator of the nonselective cation current induced by aluminum fluoride in guinea-pig chromaffin cells

Internal administration of the G protein activator, guanosine-5′- o-(3-thiotriphosphate) (GTPγS) or aluminum fluoride (AlF) complex, produce an inward nonselective cation current ( I NS) at −55 mV. This current was rapidly diminished under conditions of high intracellular Mg 2+ ([Mg 2+] = 979 μM), the half decay time ( T 1 2 ) being 80 to 100 s. As [Mg 2+] in AlF solutions decreased from 400 to 12 μM, the maximum amplitude of AlF-induced I NS became larger and the current was diminished more slowly. The AlF I NS in the presence of 12 μM Mg 2+ reversed polarity at about + 9 mV, irrespective of the extent of decline. Bath application of muscarine produced a sustained I NS was rapidly diminished. Addition of vanadate (0.5 mM) to 979 μM Mg 2+-containing AlF solution mimicked the effects of low Mg 2+ solution. Inversely, addition of alkaline phosphatase (40 units/ml) to 12 μM Mg 2+ AlF solution reproduced the effects of high Mg 2+ solution. It is suggested that AlF complex deactivates I NS through facilitating an apparent activity of Mg 2+-dependent phosphatase.

Evidence to show the phosphoprotein nature of poly(A) polymerase in germinated wheat embryos: enzyme regulation through phosphorylation

Poly(A) polymerase from wheat embryos was shown to be a phosphoprotein by labelling the enzyme in vivo with [ 32P]orthophosphate. Excised germinated wheat embryos (48 h) were incubated (12 h) in a sterile solution of sucrose (2%) containing [ 32P]orthophosphate (25 mCi) for labelling phosphoproteins. Poly(A) polymerase was then purified by ATP-Sepharose affinity chromatography. The ATP-Sepharose fraction showed a single protein-stained band on native PAGE that matched with the activity peak of the unlabelled enzyme. This proved the electrophoretic homogeneity of the enzyme preparation. Serial scanning of the parallel unstained gel showed a single 32P-labelled radioactive peak that matched the protein-stained band. Analysis of 32P-labelled purified poly(A) polymerase on SDS-PAGE also showed a single radioactive band that coincided with the protein-stained band (65 kDa) of a monomeric poly(A) polymerase. Chemical characterization of the 32P-labelled purified poly(A) polymerase by acid hydrolysis showed two radioactive spots by paper chromatography that matched authentic phosphoserine and phosphothreonine. These results suggested that wheat embryo poly(A) polymerase is phosphorylated in vivo at the serine and threonine residues. A significant amount of dephosphorylation (∼ 74%) of poly(A) polymerase was achieved by treating the 32P-labelled purified enzyme with alkaline phosphatase. Addition of alkaline phosphatase along with sodium ortho-vanadate, however, showed a meagre dephosphorylation (∼8%) of 32P-labelled poly(A) polymerase. This is because ortho-vanadate virtually blocked the activity of phosphatase (∼90%) and thus prevented the dephosphorylation of poly(A) polymerase. This enabled us to compare the biological activity of phosphorylated and substantially dephosphorylated forms of poly(A) polymerase. Thus, purified poly(A) polymerase was dephosphorylated with alkaline phosphatase and thereafter, phosphatase activity was blocked in situ by the addition of sodium ortho-vanadate. We observed a substantial decrease (∼ 60%) in poly(A) polymerase activity following treatment with alkaline phosphatase. However, poly(A) polymerase activity was not inhibited to any significant level by alkaline phosphatase in the simultaneous presence of sodium ortho-vanadate. We consider that the partially dephosphorylated form of poly(A) polymerase is less active than the phosphorylated form in wheat embryos.

Dual regulation of the n type K+ channel in Jurkat T lymphocytes by protein kinases A and C.

We have measured the activity of the n type K+ channel present in human (Jurkat) T lymphocytes using the patch clamp technique in the whole-cell configuration. We report that protein kinase A (PKA) and protein kinase C (PKC) modulate, in a dual manner, the K+ conductance in these cells. Activation of PKA decreases the amplitude of the current, as previously reported (Bastin, B., Payet, M. D., and Dupuis, G. (1990) Cell. Immunol. 128, 385-399), and this is also the case for 12-O-tetradecanoylphorbol-13-acetate-dependent activation of PKC. In contrast, inhibitors of PKC (H7, staurosporine, polymixin B, and anti-PKC antibody) increase the current amplitude. Of importance, down-regulation of PKC or its inhibition prevented the PKA-dependent inhibition of the K+ channels. Addition of alkaline phosphatase via the patch pipette increased the K+ conductance under basal conditions and reversed the inhibition produced by PKA. The dual modulation of K+ channels in Jurkat T cells is in agreement with the presence of consensus sequences in the primary structure of the n type K+ channel.

Photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts on a solid support: the influence of orthophosphate

We devised recently a method to trap intact isolated chloroplasts on a solid support consisting of membrane filters made of cellulose nitrate (Cerović et al., 1987, Plant Physiol. 84, 1249-1251). The addition of alkaline phosphatase to the reaction medium enabled continuous photosynthesis by spinach (Spinacia oleracea L.) chloroplasts to be sustained by hydrolysis of newly produced and exported triose phosphates and recycling of orthophosphate. In this system, simultaneous measurements of chlorophyll fluorescence and oxygen evolution were performed and their dependence on orthophosphate concentration was investigated. Optimal photosynthesis was obtained at a much higher initial orthophosphate concentration (2-4 mM) compared to intact chloroplasts in suspension. Secondary kinetics of chlorophyll fluorescence yield were observed and were shown to depend on the initial orthophosphate concentration.

Alkaline phosphatase promotes radioprotection and accumulation of WR-1065 in V79-171 cells incubated in medium containing WR-2721.

Addition of alkaline phosphatase and WR-2721 to culture medium containing V79-171 cells leads to production of WR-1065 and its disulphide forms in the medium, to cellular accumulation of WR-1065, and to radioprotection which correlates with cellular WR-1065 level.