Nonspecific Phosphomonoesterase Research Articles

Estimation and Correlation of Alkaline Phosphatase Enzymatic Activity in Saliva with and without Early Childhood Caries in South Indian Children: A Randomized Clinical Trial.

Early childhood caries (ECC) is a major common problem seen in children and is the most prevalent chronic disease that leads to discomfort, pain, and poor quality of life, affecting the health of children. Alkaline phosphatase (ALP) is a nonspecific phosphomonoesterase that functions through a phosphoery 1 intermediate to produce free inorganic phosphate. It has different isoenzymes produced by different cell types such as polymorphonuclear leukocytes, osteoblasts, macrophages, and fibroblasts within alveolar bone and/or salivary glands. Various studies show that higher ALP activity is related to periodontal disease and dental caries. This study aims to estimate and correlate salivary Alkaline Phosphatase enzyme activity in the saliva of children with and without ECC. A total of 50 children were included in the study, divided into two groups-caries-active and caries-free, each consisting of 25 participants. Unstimulated saliva samples were collected and subjected to a spectrophotometer for analysis. ALP enzyme activity levels were estimated and correlated between caries-active and caries-free children. The correlation between caries score and ALP activity was statistically significant, with a moderate correlation. The comparison of mean ALP activity between caries-active and caries-free groups was statistically significant. However, the comparison of ALP based on different age-groups and gender was not statistically significant. There was a statistically significant correlation between caries scores and the caries-active group. In conclusion, there is a substantial correlation between ALP enzyme levels and the severity of dental caries. An increase in ALP enzyme level is linked to a considerable rise in caries severity. Therefore, prevention may be possible with early detection. Thimmegowda U, Kuri PN. Estimation and Correlation of Alkaline Phosphatase Enzymatic Activity in Saliva with and without Early Childhood Caries in South Indian Children: A Randomized Clinical Trial. Int J Clin Pediatr Dent 2024;17(5):528-531.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100mU/mL with the limit of detection (LOD) of 1.48mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Emerging Roles of Human Prostatic Acid Phosphatase

Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed.

Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation

In the adult mammalian brain the subependymal layer of the lateral ventricles houses neural stem cells giving rise to young neurons migrating towards the olfactory bulb. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, differentiation and functional integration are poorly understood. Neural progenitors in situ express the tissue nonspecific form of alkaline phosphatase (TNAP), a cell surface-located nonspecific phosphomonoesterase capable of hydrolyzing extracellular nucleotides. To gain insight into the functional role of TNAP in cultured multipotent neural stem cells we applied a knockdown protocol using RNA interference with shRNA and retroviral infection. We show that TNAP knockdown reduces cell proliferation and differentiation into neurons or oligodendrocytes. This effect is abrogated by addition of alkaline phosphatase to the culture medium. Our results suggest that TNAP is essential for NSC proliferation and differentiation in vitro and possibly also in vivo.

Pyrococcus abyssi alkaline phosphatase: the dimer is the active form

Alkaline phosphatases (APs), E.C. 3.1.3.1, are non-specific phosphomonoesterases optimally active under alkaline conditions. They are classically known to be homodimeric metalloenzymes. This quaternary structure has been considered necessary for activity, although the relationship between quaternary structure and activity is not well understood. Recombinant Pyrococcus abyssi AP was previously isolated and characterized, appearing to have two active quaternary structures on native polyacrylamide gel electrophoresis: a monomer and a homodimer. The purpose of the present work was to determine the actual quaternary structure of P. abyssi AP in solution, by isolating each of the two quaternary forms and establishing the parameters governing the assembly and dissociation of the dimer. pH appeared to be an important parameter: in acidic media, the monomer/dimer ratio shifted towards monomer. Buffer composition also affected the quaternary structure: at the same pH, in potassium phosphate buffer, the two quaternary structures were observed, whereas in tris(hydroxymethyl)aminomethane hydrochloride buffer, only the dimer was observed. Metals bound to the enzyme were found to be involved in the stability of the quaternary structure. Indeed, the P. abyssi AP obtained upon removal of the metals was monomeric. Reactivation of the latter was achieved with variable efficiency. From these experiments, no active monomer could be isolated, leading the conclusion that the active form of P. abyssi AP is the homodimer.

Expression of Pyrococcus abyssi recombinant alkaline phosphatase: influences of Escherichia coli rare codons and secretion by the methylotrophic yeast Pichia pastoris

Alkaline phosphatases (APs) are non-specific phosphomonoesterases which are of interests for basic research as well as for practical applications. So far, Pyrococcus abyssi AP is the only AP which was isolated from a hyperthermophilic archaeon. In the present work, expression of this enzyme using mesophilic hosts was investigated. When P. abyssi AP was produced by Escherichia coli using the regulation of the T7 promoter, only 0.6 mg of recombinant enzyme per liter of culture were obtained. Using the same system with the co-expression of tRNAs related to the rare codons AGA, AGG and ATA, the expression level was 14-fold higher. The methylotrophic yeast Pichia pastoris was also used to produce P. abyssi AP. A recombinant pPICZαA was constructed in order to have the AP secreted in the culture medium. A Pc. pastoris cell line producing P. abyssi AP was studied in 1.2 l working volume fermentor. Secretion was achieved with low expression yield but the recombinant enzyme appeared more active than when produced in E. coli.

The 1.9 Å Crystal Structure of Heat-labile Shrimp Alkaline Phosphatase

Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature.Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9Å. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer.SAP shares the core, an extended β-sheet flanked by α-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of −80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly.

In vitro efficacies of phosphorolytic enzymes synthesized in mycelial cells of Aspergillus niger AbZ4 grown by a liquid surface fermentation.

Activities of phytase, a pH 6.0 optimum nonspecific phosphomonoesterase and phosphodiesterase assayed toward bis(p-nitrophenyl)phosphate (phosphodiesterase I) and against p-nitrophenylphosphorylcholine (phosphodiesterase II), were partially purified from mycelial extracts of Aspergillus niger AbZ4 cultivated on a molasses medium by a liquid surface fermentation method. After elimination of phosphate from the medium, 7.3- and 3.5-fold enhancements in specific activities of phytase and phosphodiesterase II were observed. Efficacies of mycelial protein fractions in dephosphorylating a wheat-based broiler feed were determined in vitro according to a procedure that simulated digestion in the intestinal tract of poultry. The addition of 0.052 mg of protein from fractions, each of which was high in either pH 6.0 optimum phosphomonoesterase, phosphodiesterase I, phosphodiesterase II, or phytase per gram of a feed sample resulted in the enhancement of phosphorus release by 10, 11, 27, and 88%, respectively. In the presence of an excess of commercial phytase, the addition of the mycelial fraction high in phytase increased the dephosphorylation rate by 56%. The fraction high in phosphodiesterase II enhanced feed dephosphorylation by 8% in the presence of an excess of commercial phytase and commercial acid phosphatase.

Modulation of uptake of organic cationic drugs in cultured human colon adenocarcinoma Caco-2 cells by an ecto-alkaline phosphatase activity.

Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto-ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco-2 cells, a putative modulation of 1-methyl-4-phenylpyridinium (MPP(+)) apical uptake by an ecto-ALP activity. Ecto-ALP activity and (3)H-MPP(+) uptake were evaluated in intact Caco-2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane-bound ecto-ALP expressed on the apical surface of Caco-2 cells was studied at physiological pH using p-nitrophenylphosphate as substrate. The results showed that Caco-2 cells express ALP activity, characterized by an ecto-oriented active site functional at physiological pH. Genistein (250 micro M), 3-isobutyl-1-methylxanthine (1 mM), verapamil (100 micro M), and ascorbic acid (1 mM) significantly increased ecto-ALP activity and decreased (3)H-MPP(+) apical transport in this cell line. Orthovanadate (100 micro M) showed no effect on (3)H-MPP(+) transport and on ecto-ALP activity. On the other hand, okadaic acid (310 nM) and all trans-retinoic acid (1 micro M) significantly increased (3)H-MPP(+) uptake and inhibited ecto-ALP activity. There is a negative correlation between the effect of drugs upon ecto-ALP activity and (3)H-MPP(+) apical transport (r = -0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco-2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto-ALP activity may be involved in this process.

The cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental inorganic phosphate

Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.

The cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental inorganic phosphate.

Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.

26] Alkaline phosphatase from Thennotoga neapolitana

Alkaline phosphatase (AP) (orthophosphoric-monoester phosphohydrolase) is a nonspecific phosphomonoesterase that functions through a phosphoseryl intermediate to produce free inorganic phosphate or to transfer the phosphoryl group to other alcohols. This enzyme is abundant in both prokaryotes and eukaryotes. In Escherichia coli , AP is involved in recovering phosphate from esters when free inorganic phosphate is depleted. The precise function of mammalian APs is still unclear. With a single mutation in its structural gene causing a fatal human hereditary disease—hypophosphotasia—it is postulated that AP participates in modulating bone mineralization. This chapter describes the purification procedure and the biochemical characteristics of the AP from the hyperthermophilic gram-negative eubacterium Thermotoga neapolitana and of the recombinant enzyme expressed in E. coli. Biochemical properties of this enzyme are compared to those of its calf intestine and E. coli homologs.

Distribution of Hexokinase and ATPase in the Brain of DDT-Exposed Fish

+, Mg + and K + dependent ATPase (Chetty et al. 1978), brain ATPase (Srivastava et al. 1979), G-6-p dehydrogenase (Buhler and Benville 1969), gluconeogenic enzymes and non-specific phosphomonoesterases, tissue lipids and cholesterol content in various animal species including fish (Shaffi 1982). However, the studies regarding the effect of DDT on the metabolic compartmentation of the brain was not yet investigated on any fresh water Indian fish. In the present study, an attempt has been made to study the effect of sublethal dose of DDT on differential distribution of hexokinase, Na + , K + -ATPase 2+ -ATPase in various regions of the brain in Labeo rohita (Ham), Clarias batrachus (Linn) and Channa Punctatus (Bloch) from a tropical environment on a comparative basis.

Effect of P-Glycoprotein Modulators on Alkaline Phosphatase Activity in Cultured Rat Hepatocytes

Alkaline phosphatases (orthophosphoric-monoester phosphohydrolase, E.C. 3.1.3.1) are a group of nonspecific phosphomonoesterases located primarily in the plasma membrane of the cells in which they occur [1].It was recently demonstrated that alkaline phosphatase (ALP) concentration in different tissues is positively correlated with the extent of exchange surface per unit volume of the tissue, suggesting an association between ALP and transport systems [2]. Moreover, several groups [3,4,5] obtained evidence of an involvement of ALP in the modulation of P-glycoprotein activity in hepatocytes. The aim of the present study was to determine the putative influence of compounds known to modulate P-glycoprotein-mediated transport on hepatic ALP activity, by using primary cultured rat hepatocytes.The K_m and V_max values of ALP were determined (657.2 μM (306.8-933.1) and 32.0±1.5 nmol mg protein^–1 min^–1, respectively). Vanadate and corticosterone concentration-dependently reduced ALP activity, producing maximal reductions of 79% (100 μM) and 71% (100 μM), respectively. The IC50’s were found to be 7.9 μM (2.1-29.5 μM) and 2.4 μM (0.2-35.2 μM), respectively. Cyclosporin A, verapamil, octreotide, kaempferol, daunomycin and genistein produced a concentration-dependent increase in ALP activity. ALP activity was maximally increased to 253%, 390%, 180%, 487%, 449% and 193% of control in the presence of 100 μM cyclosporin A, 50 μM verapamil, 10 μM octreotide, 100 μM kaempferol, 100 μM daunomycin and 1 μM genistein, respectively.The results show that all P-glycoprotein modulators tested were able to significantly affect the activity of hepatic-ALP. These effects on ALP activity may contribute to the modulation of P-glycoprotein activity by these drugs.

Purification and characterization of an acid phosphatase from Lactobacillus plantarum DPC2739

An acid phosphatase was partially purified from a cell-free extract of Lactobacillus plantarum DPC2739 by a combination of anion-exchange chromatography on DEAE-Sephacel, hydrophobic interaction chromatography on Phenyl Sepharose, gel permeation chromatography on Sephacryl S200 and high performance anion-exchange chromatography on MonoQ. The native enzyme (∼110 kDa) was tetrameric with a subunit molecular mass of ∼27 kDa. The enzyme was heat-stable, retaining ∼60% of its activity after heating for 30 min at 70°C. It was optimally active in the pH range 3.5–5.0 and at 40°C. The enzyme was strongly inhibited by 0.5 mM sodium fluoride, and hexametaphosphate and by 5 mM orthophosphate, tripolyphosphate and pyrophosphate. It was insensitive to metal chelators (ethylenediaminetetraacetic acid and o-phenanthroline), ascorbic acid, sulphydryl blocking agents (e.g., N-ethylmaleimide), phenylmethylsulphonyl fluoride and divalent metal ions at 5 mM concentration. The enzyme appeared to be a non-specific phosphomonoesterase and hydrolysed a number of phosphate esters. The amino acid sequence of the first 20 residues was determined and showed some homology with mammalian, yeast and Escherichia coli acid phosphatases, phosphoglycerate mutases and phosphoglycerokinases with a common motif Arg-His-Gly. ©

Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile ser102

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6×105-fold, 105-fold and 104-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Kmratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 Å. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.

Mechanism of Fe(III) – Zn(II) Purple Acid Phosphatase Based on Crystal Structures

Purple acid phosphatase is a widely distributed non-specific phosphomonoesterase. X-ray structures of the dimeric 111-kDa Fe(III)-Zn(II) kidney bean purple acid phosphatase (kbPAP) complexed with phosphate, the product of the reaction, and with tungstate, a strong inhibitor of the phosphatase activity, were determined at 2.7 and 3.0 Å resolution, respectively. Furthermore the resolution of the unligated enzyme, recently solved at 2.9 Å could be extended to 2.65 Å with completely new data. The binding of both oxoanions is not accompanied by larger conformational changes in the enzyme structure. Small movements with a maximal coordinate shift of 1 Å are only observed for the active site residues His295 and His296. In the inhibitor complex as well as in the product complex, the oxoanion binds in a bidentate bridging mode to the two metal ions, replacing two of the presumed solvent ligands present in the unligated enzyme form. As also proposed for the unligated structure a bridging hydroxide ion completes the coordination spheres of both metal ions to octahedral arrangements. All three structures reported herein support a mechanism of phosphate ester hydrolysis involving interaction of the substrate with Zn(II) followed by a nucleophilic attack on the phosphorus by an Fe(III)-coordinated hydroxide ion. The negative charge evolving at the pentacoordinated transition state is probably stabilized by interactions with the divalent zinc and the imidazole groups of His202, His295, and His296, the latter protonating the leaving alcohol group.

Human prostatic acid phosphatase: Selected properties and practical applications

Human prostatic acid phosphatase (EC 3.1.3.2) is a non-specific phosphomonoesterase, synthetized and secreted into seminal plasma under androgenic control. The enzyme is a dimer of molecular weight around 100 kDa. Gene coding this protein is localized on chromosome 3. Since many years prostatic phosphatase has been used as a marker for diagnosis and therapy control of cancer of the prostate gland. The biological role of this enzyme, however, remains unknown and needs further exploration.

Reaction mechanism of alkaline phosphatase based on crystal structures: Two-metal ion catalysis

Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E–P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn 2+ and one Mg 2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (P i), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2·0 Å resolution. Some parts of the molecule have been retraced, starting from the previous 2·8 Å study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2·5 Å resolution, and the phosphate-free native enzyme at 2·8 Å resolution. At pH 7·5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E–P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E · P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.

GPC phosphodiesterase and phosphomonoesterase activities of renal cortex and medulla of control, antidiuresis and diuresis rats

Glycerylphosphorylcholine (GPC) concentration was reported to be elevated in renal medulla of experimental animals deprived of water. The activities of GPC phosphodiesterases were similar in homogenates and membrane subfractions of renal cortex prepared from control, diuresis and antidiuresis rats. There were no differences in these preparations' ability to hydrolyze phosphorylcholine. In contrast, there was a nearly 50% reduction of non-specific phosphomonoesterase activity, using p-nitrophenylphosphate as substrate and membrane subfractions prepared from the antidiuresis animals. It is suggested that as a consequence, a pathway for the formation from L-α-glycerylphosphate is activated.