Blood appearance of rat alkaline phosphatase originating from the duodenum in vitro

The major source of rat serum alkaline phosphatase (ALP) is well known to be from the intestinal enzyme, but it is still unclear whether it is from the duodenal or the ileal enzyme. The organic origin was investigated by means of two-dimensional electrophoresis. Major isoelectric points and molecular masses for activities of duodenal enzyme treated with both phosphatidylinositol-specific phospholipase C and neuraminidase were identified apparently with those of the major serum enzyme. In organ culture, the normal duodenal enzyme was released in the highest amounts to the culture medium. These results indicate that the major source of serum ALP in adult rats is basically from the duodenal enzyme. On the other hand, lectin affinity chromatography for ALPs showed that the ALP in the medium from culture duodenum and liver had the same complex-type sugar chain as with the ALP in the duodenal tissue. Although the duodenal ALP induced by glucosamine in vitro had the hybrid-type chain, sugar chains of the induced ALP in the culture medium were of the complex type, indicating that medial ALPs possessing the same sugar chain as the native duodenal enzyme, complex type, are mainly released from their tissues in normal conditions.     

Simultaneous microanalysis of N-linked oligosaccharides in a glycoprotein using microbore graphitized carbon column liquid chromatography-mass spectrometry

We previously reported that graphitized carbon column liquid chromatography-mass spectrometry (GCC-LC-MS) is very useful for the structural analysis of carbohydrates in a glycoprotein. In this study, GCC-LC-MS was adapted for the simultaneous microanalysis of oligosaccharides. A variety of oligosaccharide alditols prepared from fetuin, ribonuclease B, and recombinant human erythropoietin were used as model oligosaccharides. The use of microbore GCC-LC-MS was found to be successful for rapid, sensitive, and simultaneous analysis of high-mannose-type, desialylated fucosyl complex-type, sialylated complex-type, and sialylated fucosyl complex-type oligosaccharide alditols. Furthermore, we demonstrate that this method is applicable to the analysis of carbohydrate heterogeneity in a glycoprotein that possesses diverse oligosaccharides. Microbore GCC-LC-MS was able to characterize high-mannose-type, hybrid-type, and complex-type oligosaccharides in tissue plasminogen activator produced from human melanoma cells in a single analysis.     

Exploration of oligosaccharide-protein interactions in glycoprotein quality control by synthetic approaches

High-mannose-type oligosaccharides, which are cotranslationally introduced to nascent polypeptides, play important roles in glycoprotein quality control. This process is highly complex, involving a number of lectins, chaperones, and glycan-processing enzymes. For example, calnexin and calreticulin (CRT) are molecular chaperones that recognize monoglucosylated forms of high-mannose-type glycans. UDP-glucose : glycoprotein glucosyltransferase (UGGT) only glucosylates high-mannose-type glycans attached to partially folded proteins. Fbs1 is a component of ubiquitin ligase that recognizes sugar chains. Although recent studies have clarified the properties of these proteins, most of them used oligosaccharides derived from natural sources, which contain structural heterogeneity. In order to gain a more precise understanding, we started our program to comprehensively synthesize high-mannose-type glycans associated with a protein quality control system. Additionally, investigation of artificial glycoproteins led us to the discovery of the first nonpeptidic substrate of UGGT. These synthetic oligosaccharide probes have allowed us to conduct quantitative evaluations of the activity and specificity of CRT, Fbs1, and UGGT.     

Synthesis and Langmuir studies of bivalent and monovalent alpha-D-mannopyranosides with lectin Con A

Highly avid interaction between carbohydrate ligands and lectin receptors nominally requires the ligand presentation in a clustered form. We present herein an approach involving Langmuir monolayer formation of the sugar ligands and the assessment of their lectin binding at the air-water interface. Bivalent alpha-D-mannopyranoside containing the glycolipid ligand was used to study its binding profiles with lectin Con A, in comparison to the corresponding monovalent glycolipid. In addition to the bivalent and monovalent nature of the glycolipid ligands at the molecular level, the ligand densities at the monolayer level were varied with the aid of a nonsugar lipid molecule so as to obtain mixed monolayers with various sugar-nonsugar ratios. Lectin binding of bivalent and monovalent ligands at different ratios was monitored by differential changes in the surface area per molecule of the mixed monolayer, with and without the lectin. The present study shows that maximal binding of the lectin to the bivalent ligand occurs at lower sugar densities at the interface ( approximately 10% sugar in the mixed monolayer) than for that of the monovalent ligand ( approximately 20% sugar in the mixed monolayer). It is observed that complete coverage of the monolayer with only the sugar ligands does not allow all of the sugars to be functionally active.     

Design and synthesis of fluorogenic substrates that target protein phosphatases

We have successfully designed and synthesized new fluorogenic probes that specifically target different classes of protein phosphatases. The fluorescence profiles of the probes have been studied using 12 different phosphatases, and results showed that, besides alkaline and tyrosine phosphatases, our probes were able to detect serine/threonine as well as acid phosphatases.     

Analysis of enzymatically glucosylated flavonoids by capillary electrophoresis

HPCE with UV detection was applied to the analyses of enzymatically glucosylated flavonoids, which are used as natural food additives in Japan. Four items, which have flavonol or flavanone as aglycone, were analyzed. Each of these items is a mixture of glycosides with various lengths of maltooligosaccharide chain. On capillary zone electrophoresis with an untreated fused-silica capillary at alkaline pH, glycosides with longer sugar chains migrated more rapidly. Flavonol glycosides with 1-13 glucose units were distinguished with the borate buffer (pH 10.0). Flavanone glycosides needed higher pH values for good separation than flavonol glycosides.     

Elastic Properties of Semi-Flexible Chains and Networks

Summary: Elastic properties of noncharged polymers of stiffness ranging from flexible to rigid chains are determined from Monte Carlo simulations. The discrete wormlike chain (WLC) model with self-interacting units is applied to chains of intermediate lengths. Elastic free energy and the force-extension profiles of chains of variable stiffness are computed in an isometric ensemble. Occurrence of a plateau on the force-extension curves at intermediate chain stiffness is noted. Qualitative differences are found between force profiles from simulations and from the standard (ideal) WLC model. The single-chain data on influence of bending stiffness were employed in the three-chain model of networks. Stress-strain relations for networks show a highly nonlinear behavior with the marked strain-stiffening effect.     

Soft effective interactions between weakly charged polyelectrolyte chains

We apply extensive molecular dynamics simulations and analytical considerations in order to study the conformations and the effective interactions between weakly charged, flexible polyelectrolyte chains in salt-free conditions. We focus on charging fractions lying below 20%, for which case there is no Manning condensation of counterions and the latter can be thus partitioned in two states: those that are trapped within the region of the flexible chain and the ones that are free in the solution. We examine the partition of counterions in these two states, the chain sizes and the monomer distributions for various chain lengths, finding that the monomer density follows a Gaussian shape. We calculate the effective interaction between the centers of mass of two interacting chains, under the assumption that the chains can be modeled as two overlapping Gaussian charge profiles. The analytical calculations are compared with measurements from molecular dynamics simulations. Good quantitative agreement is found for charging fractions below 10%, where the chains assume coil-like configurations, whereas deviations develop for charge fraction of 20%, in which case a conformational transition of the chain towards a rodlike configuration starts to take place.     

Comprehensive synthesis of ER related high-mannose-type sugar chains by convergent strategy

Systematic synthesis of high-mannose-type sugar chains of asparagine-linked glycoproteins is described. To construct the target sugar chains, we employed the convergent route, using three oligosaccharide components, the common hexasaccharide, branched tri-, tetra- and pentasaccharides, and mono-, di-, and triglucosyl fragments. Construction of the β-mannoside linkage was performed using p-methoxybenzyl-assisted intramolecular aglycon delivery. The hexasaccharide fragment was coupled with the branched mannooligosaccharide donors such as M5, M4B, M4C, and M3 to give undecasaccharide (M9), decasaccharide (M8B and M8C), and nonasaccharide (M7), respectively. Incorporation of mono-, di-, and triglucosyl fragments toward them gave tetradecasaccharide (G3M9), tridecasaccharide (G2M9), dodecasaccharide (G1M9), undecasaccharide (G1M8B and G1M8C), and decasaccharide (G1M7), respectively.     

Effects of sample preparation on methylmercury concentrations in Arctic organisms

The biogeochemical cycling of mercury (Hg) in the marine environment is an issue of global concern, as consumption of marine fish is a major route of human exposure to the toxic specie methylmercury (MeHg). The most widely utilised and accepted technique for preparing biological tissue samples for the analysis of MeHg involves an alkaline digestion of the sample. Recent studies suggest, however, that this technique is inadequate to produce satisfactory recoveries for certain biological samples, including fish, fur, feathers and other ‘indicator’ tissues which contain relatively high levels of MeHg. Thus an improved acidic extraction method has been proven to produce more satisfactory results for a wide range of biological tissues. The present study compares the two methods on real sample material from different organisms of an Arctic marine food chain, and shows how this could lead to misinterpretation of analytical results. Results show significantly (p < 0.05) lower concentrations for alkaline digestion for large parts of the food chain; especially in fish and birds. The mean differences in concentrations found between the two different methods were 28, 31 and 25% for fish (Polar and Atlantic cod), Little Auk and Kittiwake, respectively. For samples lower in the food chain (i.e. zooplankton and krill) no significant differences were found. This leads to a clear underestimation of the levels of MeHg found higher up in these food chains; the ratio of MeHg to Hg in biological samples; and thus potentially erroneous conclusions drawn from these results concerning the biological cycling of mercury species. We hypothesise that the main reasons for these differences are poor extraction efficiency and/or matrix effects on the ethylation step prior to analysis. This is the first study to examine the effects of these artefacts on real environmental samples covering a complete food chain.     

Stable Sugar‐Chain‐Immobilized Fluorescent Nanoparticles for Probing Lectin and Cells

Sugar chains are important molecules in cellular recognition and signaling, and quantum dots (QDs) are a very powerful tool for in vitro and in vivo imaging. Herein, we report the preparation of stable sugar‐chain‐immobilized fluorescent nanoparticles (SFNPs) and their application to the analysis of sugar‐chain–protein interactions and cellular imaging. SFNPs were easily prepared by mixing CdTe/CdS core/shell QDs with our previously developed sugar‐chain–ligand conjugates. The obtained SFNPs were very stable and could be stored for several months. In the binding analysis, β‐galactose‐ and α‐glucose‐immobilized SFNPs were specifically interacted with Ricinus communis agglutinin I and concanavalin A, respectively, and made into aggregates. The binding interaction was detected visually, fluorescently, or both. In the experiment for cellular imaging, it was found that SFNPs were predominantly taken up by human hepatocyto carcinoma cells (HepG2), suggesting the possible usage of our designed SFNPs for various biochemical analyses of sugar chains.     

3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10(-16) mol (injected amount) with helium-cadmium laser induced detection.     

CONVEX CURVATURES OF ALKALINE ELUTION PROFILES OF DNA FROM HUMAN CELLS IRRADIATED WITH 405 nm UVA: EVIDENCE FOR INDUCTION OF SLOWLY DEVELOPING ALKALI-LABILE SITES

The alkaline (pH 12.1) elution profiles of DNA from human P3 cells exposed to monochromatic 405 nm UVA radiation deviate from exponential: on a logarithmic plot of eluted fraction of DNA vs time of elution, the rate of elution accelerates for the first 6 h. Following this period, the profiles become exponential. In contrast, the elution profiles of DNA after 520 nm green light or ionizing radiation exposures (x- and gamma rays, and fission spectrum neutrons) are always strictly exponential, evidence that the convex profiles were not due to an artifact caused by elution technique. Holding the DNA at pH 12.1 for 6 h after 405-nm exposures before initiating elution resulted in profiles that were close to exponential, with slopes similar to the final slopes observed following the 6-h elution period in the original experiments. This is evidence that some DNA breaks develop slowly during the first 6 h of elution, as a result of exposure to alkali. Therefore, the DNA lesions induced by 405-nm light as measured by the alkaline elution technique are apparently heterogeneous and include a major class of alkali-labile sites that develop slowly during incubation at pH 12.1. Convex profiles also occur following exposure of the cells to visible light at 434 and 512 nm.     

Efficient charge transport along phenylene-vinylene molecular wires

We have studied the motion of charge carriers along isolated phenylene-vinylene (PV) chains using a combination of experimental and theoretical methods. The conductive properties of positive charges along PV chains in dilute solution were studied by using the pulse-radiolysis time-resolved microwave conductivity (TRMC) technique. This technique enables the measurement of high-frequency (tens of GHz) charge carrier mobilities along isolated PV chains without the use of electrodes. The charge carrier mobility along PV chains with finite and infinite length was studied theoretically by charge transport simulations with parameters from density functional theory (DFT) calculations. The high-frequency charge carrier mobility is found to depend strongly on the conjugation length of the PV chains and is found to increase both with increasing length of the PV chain and with increasing conjugation fraction. The experimental results are in good agreement with the calculated results. On the basis of this combined experimental and theoretical study an intrachain charge carrier mobility of a few tens of cm2/Vs is expected for an infinitely long PV chain without conjugation breaks.     

Voltammetric detection of lectin using sugar labeled with electroactive substance.

The voltammetric detection of soybean agglutinin (SBA) was investigated on the basis of an interaction between the lectin and a sugar. Because galactose and lactose combined with SBA, the sugars were labeled by a Schiff base with an electroactive daunomycin. After the labeled sugar and SBA were mixed, measurements were carried out by voltammetry. When SBA-sugar binding occurs, a part of daunomycin of the labeled sugar is taken to the binding sites. As a result, SBA is detected by a change in the peak current of daunomycin, and the SBA-sugar interaction is evaluated. The length of the alkyl chain between daunomycin and the sugar was also considered. The electrode response to the concentration of SBA was linear over the range of 0.04-0.8 microg min(-1). The merits of this procedure are the convenient preparation of labeled sugar and a rapid measurement without separation. On the other hand, the detection of sugar at the 10(-9) mol dm(-3) level was achieved by a competitive reaction to limited binding sites of the lectin between the sugar and the labeled sugar.     

Determination of Zinc(II) by the Reactivation of the Apoenzymes of Alkaline Phosphatases from Diverse Sources

ABSTRACT

A sensitive and selective method for the determination of Zn(II) based on the reactivation of the apoenzymes of alkaline phosphatases from diverse sources: chicken intestine, seal slim intestine and Escherichia coli, has been developed. To obtain the apoenzymes, EDTA was selected as an effective inhibitor of alkaline phosphatases. In the presence of EDTA excess in the solution, the pseudo apoenzymes were obtained. Dialysis of the enzymes against the chelating agent resulted in obtaining inactive zinc-free apo-phosphatases (true apoenzymes). The reactivation of the pseudo- and true apoenzymes of alkaline phosphatases from various sources by zinc(II) was applied for its determination. The proposed procedures are highly sensitive (limit detection of Zn(II) = 0.3-0.4 ng ml^?1), selective (no other metals reactivate the apophosphatases from seal slim intestine and Escherichia coli), accurate, simple and rapid. The schemes of the mechanism of the reactivation effects are discussed.     

Convenient analytical methods for endo-type glycosidase that acts on glycoconjugates and their application in glycotechnology.

The following procedures were established in order to develop useful degradation enzymes of glycoconjugate for developing postgenome and postproteome research: (1) Enzyme activity with a short time reliability was measured using small amounts by HPLC. (2) The structures of the sugar chains liberated from the glycoconjugate were non-destructively analyzed using small amounts of sugar chains only by 1D 1H-NMR and H-H COSY spectrometry and a computer simulation of the spectrum. (3) The conformations of the sugar chains liberated from a glycoconjugate in aqueous solution were estimated using 1D 1H-NMR and H-H COSY spectrometry and the anisotropic effect. Endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 developed using the above methods transferred the sugar chain to sugars and peptides; therefore, it was also an effective enzyme when synthesizing sugar chains and glycopeptides.     

Lectin affinity electrophoresis of alkaline phosphatase for the differentiation of bone and hepatobiliary disease

An affinity electrophoresis procedure is described for the separation and quantification of the bone- and liver-derived fractions of alkaline phosphatase in plasma. Separation is carried out on cellulose acetate membrane pre-soaked with buffer containing wheat germ lectin. The electrophoretic mobility of the bone enzyme is preferentially retarded by the lectin and this fraction is well separated from the liver fraction. After separation, enzyme activity is demonstrated by staining using an indigogenic alkaline phosphatase substrate incorporated in agar gel, and the stained fractions quantified by densitometry. The procedure has low imprecision, good linearity, and the activities of the bone and liver fractions correlate well with values obtained using nonelectrophoretic quantification methods. The procedure is especially suitable for use in the diagnostic laboratory.     

Partitioning of Semiflexible Macromolecules into a Slit in Good Solvents

We investigate by Monte Carlo simulations the partitioning of semiflexible chains into slits the sizes of which are in the range of coil dimensions. The investigated chains have variable rigidities within the coil regime not reaching the rigid rod limit. Noticeable deviations of the commonly used approximate persistence length from its exact counterpart are reported. The partitioning of semiflexible chains in the reduced plot of partitioning coefficient versus confinement is located between the results for the partitioning of a sphere and for a rigid rod. At large confinement, and for the most rigid chains investigated, the scaling law for partitioning approaches that of the rigid rods. We advocate presenting results based both on the reduced and absolute plots for drawing the correct conclusions. On increasing concentration, it is apparent that the differences in partitioning resulting from variable chain rigidity appear only in the dilute solution. At higher concentrations the differences vanish. The weak‐to‐strong penetration transition on an increase of concentration is explained using the scaling approach by the change of the mobility unit from the coil dimension to a concentration correlation length, similarly to that of flexible chains. The microscopic picture of partitioning represented by various concentration profiles in the slit leads to the conclusion that stiffer chains are able to fill the depletion layer at the walls more readily.     

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.