Calf Intestinal Alkaline Phosphatase I. Improved Isolation Method and Molar Composition of the Purified Phosphatase

A modified and improved method for isolation of calf intestinal alkaline phosphatase is described. By this method 300 to 400 mg of pure enzyme was prepared in a relatively short time. On the basis of the results of ultracentrifugation and of the free, polyacrylamide and immunoelectrophoresis the phosphatase obtained is found to be a homogenous glycoprotein, containing firmly bound zinc, magnesium and phosphoric acid. The molar composition of the enzyme and the catalytic activity were determined with different substrates and buffers.     

Acid pyrophosphatase from red kidney beans

Partial purification of acid pyrophosphatase activity from dried red kidney beans was achieved. The crude enzyme was found to adhere to plastic and was very unstable. These problems were solved by extraction with low pH and high-ionic-strength buffers. This extraction procedure separated acid pyrophosphatase activity into three parts. One of these activities appears to correspond to the purple phosphatase isolated by other workers (1—3). The other two fractions showed both general phosphomonoesterase and pyrophosphatase activity, but were most active with pyrophosphate and were used for further characterization. The pH optimum for the enzyme was approx 5.5-6.0 with pyrophosphatase, and it exhibited substrate inhibition with pyrophosphate and ATP at low pH. The partially purified acid pyrophosphatase was estimated to be a dimer of approx 98 kDa (mol wt estimated by gel filtration on Sephacryl S-200) with no detectable carbohydrate or iron content. Of the cations tested for their effect on pyrophosphatase activity, iron was the most inhibitory, followed by magnesium and zinc.     

An enzymatic method for determining magnesium

The effect of magnesium ions on the catalytic activity of alkaline phosphatases from three different sources in the reaction of p-nitrophenyl phosphate hydrolysis was studied. It was found that magnesium in concentrations of 0.6 ng/mL-20 µg/mL significantly activated alkaline phosphatase from chicken intestine; in higher concentrations (0.02–0.2 mg/mL), it weakly activated the enzyme from E. coli and had no effect on the catalytic activity of the enzyme from the small intestine of the Greenland seal. The strongest activating effect was observed in a Tris-HCl buffer solution at pH 9.8. The activating effect of magnesium on alkaline phosphatase from chicken intestine was used as the basis for developing a highly sensitive and selective enzymatic procedure for determining magnesium (0.6–6.0 ng/mL; RSD = 4% at c _min; n = 5) by spectrophotometrically monitoring the rate of enzymatic reaction. The developed procedure was applied to the determination of magnesium in urine.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 4, 2005, pp. 425–433.Original Russian Text Copyright © 2005 by Zhavoronkova, Muginova, Shekhovtsova.     

The Calf Intestinal Alkaline Phosphatase. II. Reaction between the metal content and the enzyme activity

Pure alkaline phosphatase (EC 3.1.3.1; 1500 U/mg) was dialyzed at 4° during 168 h against water, 10^?4M EDTA or 10^?4M o-phenanthroline. During the dialysis, samples were periodically removed and analyzed for metal content and activity. The results indicate that 1 mol of native calf intestinal alkaline phosphatase contains 4 g-atom of zinc and 4 g-atom of magnesium tightly bound, and that both metal ions are necessary for full enzyme activity. The dialyzed, partially demetallized enzyme could be reactivated by the addition of zinc and/or magnesium salts.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium.

The alkaline phosphatase present on isolated brush border and basal lateral membranes of rat duodenal epithelium were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6--9.8, similar substrate km's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin of papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000--150,000, and both enzymes show an Rf value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The Vmax of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 mumoles/mg-h as opposed to 14 mumoles/mg-h. The differences in Vmax are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Evidence against in vitro modulation of rat liver cholesterol 7 alpha-hydroxylase activity by phosphorylation-dephosphorylation: comparison with hydroxymethylglutaryl CoA reductase

The activity of cholesterol 7 alpha-hydroxylase in rat liver microsomes was investigated under conditions favourable for phosphorylation-dephosphorylation. The enzyme activity was similar in the presence or absence of sodium fluoride during preparation. Preincubation with ATP and magnesium did not affect the enzyme activity. Cholesterol 7 alpha-hydroxylase was inhibited by alkaline phosphatase, but this inhibition was similar also after inactivation of the phosphatase. Under similar conditions, rat hepatic hydroxymethylglutaryl CoA reductase activity was clearly modulated in agreement with phosphorylation-dephosphorylation. The absence of such a modulation of cholesterol 7 alpha-hydroxylase argues against involvement of phosphorylation-dephosphorylation in the regulation of this enzyme.     

麦芽酸性磷酸酶的化学修饰及紫外光谱

本文研究化学修饰剂2,3-丁二酮(Butanedione)、β-巯基乙醇(Mercaptoethanol)、对硝基苯磺酰氟(4-Nitrobenzensulfonyl fluoride,NBSF)和焦碳酸二乙酯(Diethyl pyrocarhonate,DEPC)在不同浓度、不同时间条件下对麦芽酸性磷酸酶(ACPase,E.C.3.1.3.2)相互作用后的活力及紫外光谱的变化。结果表明:色氨酸和精氨酸可能是维系麦芽酸性磷酸酶催化功能的必需基团,酶分子中二硫键和组氨酸残基不参与该酶分子活性中心的构成。     

pH-metric detection of alkaline phosphatase activity as a novel biosensing platform

A detection of alkaline phosphatase (ALP, EC 3.1.3.1) activity by the monitoring of pH changes caused by the biocatalytic action of the enzyme has been experimentally examined. Enzymatically catalyzed hydrolysis of monofluorophosphate has been found to be the best basis for such measurements. Protolytic equilibria connected with the developed biosensing system were recognized and the optimal conditions for the assay have been found. Advantages and disadvantages of the developed (bio)sensing scheme have been discussed. The prototype of pH-ALP based enzyme electrode has been demonstrated. Potential utility of such substrate-enzyme-sensor system for the development of a new group of biosensors has been announced.     

Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.     

Adsorption, desorption and activities of acid phosphatase on various colloidal particles from an Ultisol

Adsorption, desorption and activity of acid phosphatase on various soil colloidal particles and pure clay minerals were studied. Higher adsorption amounts and low percentage of desorption of acid phosphatase were found on fine soil clays (<0.2 μm). Electrostatic force and ligand exchange are the major driving forces that are involved in the adsorption of enzymes on soil clays. More enzyme molecules were adsorbed on soil clays in the presence of organic components. However, enzymes on organic clays were more easily released. One-third of the enzyme on goethite was adsorbed via ligand exchange process. Some other interactions, such as van der Waals force, hydrophobic force and hydrogen bonding may be more important in the adsorption of enzyme on kaolinite and the enzyme in this system cannot be easily removed. Coarse clays (0.2–2 μm) and inorganic soil clays had higher affinities for enzyme molecules than fine clays and organic clays, respectively. The activity of enzyme bound on soil clays was inhibited and the thermal stability was increased in the presence of organic matter. Data obtained in this study are helpful for a better understanding of the interactions of enzymes with inorganic and organic constituents in soil and associated environments.     

Liquid Chromatography Tandem Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy of Magnesium (II) Gluconate Solution

Magnesium gluconate is a classical pharmaceutical compound used as a source of magnesium for the prevention and treatment of hypomagnesemia. To the best of our knowledge, a robust and reliable liquid chromatography tandem mass spectrometry technique has not yet been reported for the qualitative and quantitative analysis of magnesium gluconate. This study describes the method development for the LC–ESI–MS/MS analysis of magnesium gluconate using three different reversed-phase HPLC conditions (Method I–III) with comprehensive fragmentation pattern and the structural characterization by NMR spectroscopy. The LC–MS and NMR data were found in accordance with the structure of magnesium gluconate. When magnesium gluconate was dissolved in the acetonitrile and water–methanol solutions, it exists in situ in three different forms: magnesium gluconate itself, gluconic acid, and magnesium gluconate chelate with gluconic acid by a coordinate covalent bond. Method I exhibited pseudo-molecular ion peaks with more magnesium gluconate chelates with gluconic acid, while method II showed an adduct of magnesium gluconate with the solvent along with the molecular ion peak. There was no pseudo-molecular ion peaks found in method III. Thus, method III was found to be the more accurate, robust and reliable LC–MS method for the qualitative and quantitative analysis, structural characterization, and could also be suitable for the pharmacokinetic study of magnesium gluconate. The detailed fragmentation analysis might be useful for the structural characterization of unknown divalent organometallics.     

Microfabricated bioreactor chips for immobilised enzyme assays

A microfabricated device is reported that has been designed to permit the in situ packing of a section of channel with enzyme immobilised onto controlled pore glass (CPG). It is fabricated from glass and polydimethylsiloxane and to prevent dead volumes, has dedicated channels for packing the reactor. The device has the advantage of being simple in design, the flow through enzyme reactor channel being simply a widened section of the analyte channel. The system is suitable for both hydrodynamic and electro-osmotic pumping, and is designed such that when the packing is exhausted it can be repacked. Controlled pore glass provides a reproducible none swelling, high porosity medium onto which the enzyme could be immobilised. The large particle size meant that it was vital to optimise the immobilisation procedure in order to achieve acceptable enzyme activity. The microfabricated device was developed with two enzymes of different molecular masses; alkaline phosphatase and xanthine oxidase. The pore size of the CPG was found to be very important for xanthine oxidase, where the 697 Å pore size (120-200 mesh) CPG was found to give the highest activity (18-20% activity retained after immobilisation). The microfabricated device was used for the assay of p-nitrophenyl phosphate and hypoxanthine with spectrophotometric detection at 405 and 470 nm, respectively. The limits of detection were 5 and 8 μM, respectively.     

Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT-isooctane-water reverse micelles

The kinetics of 1-naphthyl phosphate and phenyl phosphate hydrolysis, catalyzed by human prostatic acid phosphatase (PAP) entrapped in AOT-isooctane-water reverse micelles, has been studied over surfactant hydration degree (w0) range 5 to 35. Continuous spectrophotometric acid phosphatase assays, previously prepared, were employed. PAP was catalytically active over the whole w0 studied range. In order to determine steady-state reaction constants the experimental data were fitted to Hill rate equation. Positive cooperativity in substrate binding was observed, as it was earlier found in aqueous solutions. The extent of cooperativity (expressed as the value of the Hill cooperation coefficient h) increased from 1 to 4, when the micellar water-pool size was growing, at fixed enzyme concentration. In the plots of catalytic activity (kcat) versus w0, the maxima have been found at w0=10 (pH 5.6) and 23 (pH 3.8). It is suggested that catalytically active monomeric and dimeric PAP forms are entrapped in reverse micelles of w0=10 and 23, respectively.     

A Molecular Description of Acid Phosphatase

Acid phosphatase is ubiquitous in distribution in various organisms. Although it catalyzes simple hydrolytic reactions, it is considered as an interesting enzyme in biological systems due to its involvement in different physiological activities. However, earlier reviews on acid phosphatase reveal some fragmentary information and do not give a holistic view on this enzyme. So, the present review summarizes studies on biochemical properties, structure, catalytic mechanism, and applications of acid phosphatase. Recent advancement of acid phosphatase in agricultural and clinical fields is emphasized where it is presented as potent agent for sustainable agricultural practices and diagnostic marker in bone metabolic disorders. Also, its significance in prostate cancer therapies as a therapeutic target has been discussed. At the end, current studies and prospects of immobilized acid phosphatase are included.     

Characterization and Activity Determination of the Human Protein Phosphatase 2A Catalytic Subunit α Expressed in Insect Larvae

Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit ?? of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit ?? of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250???g per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94???mol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2?months at ?20?°C.     

Polyacrylamide gradient electrophoresis for protein purification on the milligram scale.

A preparative-scale electrophoretic technique for protein fractionation and elution on a discontinuous gradient of acrylamide is described, which permits the separation and elution of a pure protein from a mixture containing 4-20 electrophoretically different proteins. The sharpness of the gradient electrophoretic resolution is demonstrated by the separation of proteins consisting of bovine serum albumin polymers and lactate dehydrogenase and enzymes such as acid phosphatase. The compositions of various discontinuous gradients of acrylamide and their application to enzyme purification are discussed. It was found that 60% of the enzyme activity loaded on the gel is recovered after gel fractionation and elution.     

An electrochemical amplification immunoassay using bi-electrode signal transduction system

An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bi-electrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30 min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity.     

Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline

An analytical method for studying enzyme inhibition has been developed using capillary electrophoresis with laser-induced fluorescence detection. This technique is based on electrophoretic mixing of zones of enzyme and inhibitor in substrate-filled capillaries. Enzyme catalytic activity is measured by detecting the fluorescent reaction product as it migrates past the detector. Reversible enzyme inhibition is indicated by a transient decrease in product formation. The enzyme, alkaline phosphatase, has been studied using the fluorogenic substrate AttoPhos ([2,2'-bibenzothiazol]-6-hydroxy-benzthiazole phosphate). This assay has been used to quantify theophylline, a noncompetitive, reversible inhibitor of alkaline phosphatase. The detection limit for theophylline is estimated at 3 microM, and 8.6 amole of alkaline phosphatase are required for each assay. The calculated K(i) for theophylline is 90 microM for the capillary electrophoretic enzyme-inhibitor assays.     

Silica gel thin-layer chromatography of acidic phospholipids. II. Chromatographic behaviour of phosphatidylserine and phosphatidic acid applied with different cation composition on adsorbents either free of metal ions or containing a surplus of divalent metal ions.

Different salt forms of phosphatidylserine and phosphatidic acid (two acidic phospholipids) have been subjected to thin-layer chromatography on two commonly used silica adsorbents, one of which (silica gel HR) is practically free of metal ions and the other (silica gel G) contains 13% of calcium sulphate as binder. The chromatographic behaviour was studied in an acidic, a neutral and a basic solvent. Both adsorbents provided usable systems for phosphatidylserine with each of the three solvents, except for silica gel G with the neutral solvent, in which system tailing was prominent. The inclusion of calcium sulphate in the silica gel tended to impair chromatography of phosphatidylserine in acidic and neutral solvents, but improved its chromatography in the basic solvent. In all the systems, the migration was independent of the cation composition of the applied phosphatidylserine samples. For the chromatography of phosphatidic acid, only three of the systems tested were usable, and in those three, the chromatographic behaviour was independent of the cation composition of the samples. The calcium sulphate in an adsorbent increased tailing of phosphatidic acid in acidic and neutral solvents, as it did for phosphatidylserine, whereas with the basic solvent, calcium sulphate in the adsorbent caused phosphatidic acid to remain at the origin. Two one-dimensional thin-layer chromatographic systems previously recommended for the chromatography of acidic phospholipids were unsuitable for the chromatography of phosphatidic acid under the conditions used here. For both phosphatidylserine and phosphatidic acid chromatographed in acidic systems, the solvent must contain water in addition to acetic acid if excessive tailing is to be avoided.     

Structural characterization of cardiolipin by tandem quadrupole and multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization

We report negative-ion electrospray tandem mass spectrometric methods for structural characterization of cardiolipin (CL), a four-acyl-chain phospholipid containing two distinct phosphatidyl moieties, of which structural assignment of the fatty acid residues attached to the glycerol backbones performed by low-energy CAD tandem mass spectrometry has not been previously described. The low-energy MS2-spectra of the [M - H]- and [M - 2H]2- ions obtained with ion-trap or with tandem quadrupole instrument combined with ion-trap MS3-spectra or with source CAD product-ion spectra provide complete structural information for CL characterization. The MS2-spectra of the [M - H]- ions contain two sets of prominent fragment ions that comprise a phosphatidic acid, a dehydrated phosphatidylglycerol, and a (phosphatidic acid + 136) anion. The substantial differences in the abundances of the two distinct phosphatidic anions observed in the MS2-spectra of the [M -H]- ions lead to the assignment of the phosphatidyl moieties attached to the 1' or 3' position of central glycerol. Upon further collisional dissociation, the MS3-spectra of the phosphatidic anions provide information to identify the fatty acyl substituents and their position in the glycerol backbone. The MS2-spectra of the [M - 2H]2- ions obtained with TSQ or ITMS contain complementary information to confirm structural assignment. The applications of the above methods in the differentiation of cardiolipin isomers and in the identification of complex cardiolipin species consisting of multiple molecular structures are also demonstrated.