Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High-performance liquid chromatography using pellicular quaternary amine-bonded resins was used to separate a variety of neutral, sialylated, and phosphorylated oligosaccharides. At pH 4.6, sialylated compounds were separated according to number of negative charges, sialic acid linkage [alpha(2,3) compared to alpha(2,6)], and position of sialic acid linkage along a linear saccharide chain. At pH 13, the neutral sugar portion of the sialylated chain had a significant effect on the separation, due to oxyanion formation. Specifically, sialylated tetrasaccharides containing the Gal beta(1,3)GlcNAc sequence were retained much more than their Gal beta(1,4)GlcNAc- or Gal-beta(1,4)GalNAc-sialylated counterparts. Linear phosphorylated oligosaccharides could be completely separated according to number of charges and net carbohydrate content. Partial separation of linear-chain positional isomers, differing in either location of Man-6-PO4 in the chain or linkage position of Man or Man-6-PO4, was accomplished. Branched-chain phosphorylated compounds could be completely separated according to which antennae contained the Man-6-PO4. The electrochemical current generated by oxidation of sialylated, phosphorylated, and neutral oligosaccharides was compared to that of a glucose. The relative molar response factors for neutral, sialylated, and phosphorylated oligosaccharides ranged from 0.2 to 3.2. Neutral oligosaccharides gave the following molar responses for each group of structurally related compounds: (1) mono- and disaccharide, 1-1.3; (2) linear tri- and tetrasaccharides, 1.5-2.0; and (3) branched pentasaccharide-nonasaccharides, 2.4-3.1. Response factors for the sialyated compounds were not as consistent and were affected by linkage position of sialic acid. For oligosaccharides of the same size, increasing phosphorylation resulted in a twofold decrease in response factor for each added phosphate group. Therefore, conversion of sialylated and phosphorylated oligosaccharides to their neutral counterparts, using alkaline phosphatase or neuraminidase, respectively, was required for quantitative analysis of oligosaccharide mixtures using electrochemical response. Using this approach, complete separation of the parent neutral structures was obtained, the relative proportions of the neutral species were quantified, and the amount of sialic acid released was easily determined in a neuraminidase digest.
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PMID:High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection. 323 49

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4 degrees C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li, M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 1H and 31P nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(alpha 1-3)Rha(beta 1-4)Glc(beta 1-6)Galf(beta 1-6)GalNAc(beta 1- 3)Gal(alpha 1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing betaGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.
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PMID:A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V. 336 Jul 42

Previous studies in our laboratory have shown that peanut agglutinin (PNA), a lectin specific for the disaccharide Gal beta 3GalNAc, binds to immature (cortical) thymocytes of mouse and man and not to the mature (medullary) cells. Using lectin overlay of protein blots and lectin-affinity chromatography, we have found that the major PNA-binding glycoproteins on total as well as on immature (PNA+) human thymocytes correspond to two bands of Mr 170,000 and 180,000. Another glycoprotein, of Mr 110,000, also binds PNA but to a lesser extent. All three glycoproteins contain sialic acid as demonstrated by cell surface labeling with NaIO4-NaB3H4, binding of wheat germ agglutinin, and reaction with alkaline phosphatase-hydrazide. After treatment with sialidase, binding of PNA to these glycoproteins is significantly enhanced.
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PMID:Identification of peanut agglutinin-binding glycoproteins on immature human thymocytes. 348 67

Comparing the marker enzymes horseradish peroxidase (HRP), alkaline phosphatase (AP) and beta-galactosidase (beta Gal) in IgG-coupled form with respect to their temperature-dependent kinetics over a period of 22 h the temperature of 37 degrees C warrants highest substrate turnover for all enzymes at all reaction times using fluorogens. Also applying chromogens the optimum temperature for beta Gal is 37 degrees C and depends for HRP and AP on the reaction time. The substrate turnover of HRP using ABTS as chromogen is much higher compared to the other enzymes--both related to mol enzyme (molar activity) and to gram enzyme (specific activity). The turnover decreases for all enzymes in different degrees after coupling to IgG. The turnover of fluorogenic substrates is lower for all enzymes than the turnover of chromogenic substrates but due to the more sensitive detection of fluorogenic products the detection limits for all conjugates were lowered too--especially for beta Gal-IgG by a factor of 333 compared to the colorimetric procedure. In a 2-site binding enzyme immunoassay for alpha-1-fetoprotein (AFP) the detection limit for AFP was reduced by a factor of 2 only by the fluorimetry compared to the colorimetry with all 3 marker enzymes. The HRP-IgG conjugates warranted lowest detection limits for AFP (0.5-1 microgram/1), highest analytical sensitivity (slope of standard curves) at shortest periods of substrate reaction compared to the other enzymes.
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PMID:Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase? 392 20

A glycophosphoceramide concentrate prepared from tobacco leaves was shown to contain a mixture of related lipids (Kaul, K., and Lester, R. L. (1975) Plant Physiol. 55, 120-129; Kaul, K., and Lester, R. L. (1978) Biochemistry 17, 3569-3575) with the simplest and most abundant components having the structure GlcN(+/- Ac)(alpha 1 leads to 4)GlcUA(alpha 1 leads to 2)myoinositol-1-O-phosphorylceramide (Hsieh, T. C.-Y., Kaul, K., Laine, R. A., and Lester, R. L. (1978) Biochemistry 17, 3575-3579). To determine the structure of the more complex members of this series, a mixture of oligosaccharides was prepared from a carboxyl-reduced glycophosphoceramide concentrate by alkali-catalyzed hydrolysis and alkaline phosphatase treatment. A combination of reverse-phase high pressure liquid chromatography (Wells, G. B., and Lester, R. L. (1979) Anal. Biochem. 97, 184-190), normal-phase high pressure liquid chromatography, and thin layer chromatography were used to resolve several oligosaccharides as acetylated derivatives. Products of methylation analysis, CrO3 oxidation, and deacetylation-deamination were identified using chemical ionization mass spectrometry to give the following novel structures. A major tetrasaccharide was completely characterized as Gal(alpha 1 leads to 4)GlcNAc(alpha 1 leads to 4)GlcUA(alpha 1 leads to 2)myoinositol. An additional structure of a minor tetrasaccharide was partially characterized as GlcNAc(alpha 1 leads to 4)GlcUA(alpha 1 leads to ?)myoinositol(O leads from 1 alpha)Man. These are representatives of a class of acidic glycolipids from plants, possibly analogous to the acidic gangliosides found in animal cell membranes.
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PMID:Glycophosphoceramides from plants. Purification and characterization of a novel tetrasaccharide derived from tobacco leaf glycolipids. 726 25

We have developed a eukaryotic expression vector that provides a rapid and sensitive measure of transcriptional activity modulated by general and tissue-specific regulatory motifs. The lacZ structural gene has been linked to the minimal promoter of the human liver/bone/kidney alkaline phosphatase gene. In addition, a trimerized cassette of the SV40 polyadenylation region has been placed 5' of this promoter to reduce plasmid-initiated transcripts extending through the lacZ gene that would contribute to background beta-galactosidase (beta-Gal) activity. By combining the weak promoter and the poly(A) cassette, only a very low level of lacZ activity is detected in the absence of additional regulatory sequences. Regulatory domains can be inserted into this vector via a unique Bam HI restriction site and their activity can be rapidly monitored in situ via a colorimetric 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Ga) staining protocol. Also, the activity of linked regulatory domains can be measured quantitatively by assaying beta-Gal levels in cell extracts. We show that derivatives of this vector can be used to monitor the activity of general and tissue-specific control elements and can be transactivated by a single transcription factor in cotransfection experiments.
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PMID:A sensitive lacZ-based expression vector for analyzing transcriptional control elements in eukaryotic cells. 762 23

We report the development of a solid-phase assay for the activity of the enzyme GDPFuc:Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3 fucosyltransferase (alpha 1,3FT). This enzyme generates the blood group antigen Lewis x (Lex)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-R from the acceptor Gal beta 1-4GlcNAc-R. In our method, the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (lacto-N-neotetraose, LNnT) from human milk was chemically conjugated to bovine serum albumin (BSA) to generate LNnT-BSA. As a source of alpha 1,3FT to develop the assay, we used extracts of COS7 cells created to stably express the human FucTIII and FucTIV genes, both of which have alpha 1,3FT activity. LNnT-BSA was immobilized in microtiter wells and incubated with GDPFuc and cell extracts. The Lex antigen generated by alpha 1,3FT was detected with a monoclonal IgM antibody (anti-CD15). Binding of this IgM-type antibody to product was detected by one of two methods. Method 1 was based on the binding of alkaline phosphatase-conjugated goat anti-mouse IgM. Method 2 was based on the binding of a streptavidin conjugate of the recombinant bioluminescent protein aequorin to biotinylated goat anti-mouse IgM. The alpha 1,3FT assay was linear with respect to time (0-3 h), extract added (0-40 micrograms), and was dependent on GDPFuc (20 microM optimal) and LNnT-BSA. Both methods 1 and 2 allowed measurement of alpha 1,3FT in extracts of the human cell line HL-60.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Determination of GDP-Fuc:Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3 fucosyltransferase activity by a solid-phase method. 769 85

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.
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PMID:O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans. 792 59

The distributions of the hydrolases acid and alkaline phosphatase (AP and ALP), N-acetyl-beta-D-glucosaminidase (NAG), beta-glucuronidase (beta-Gluc), beta-galactosidase (beta-Gal), non-specific esterase (UE), dipeptidylpeptidases II and IV (DPPII and DPPIV), aminopeptidases M and A (APM and APA), and gamma-glutamyltransferase (GGT) were investigated in the human, pig and Lewis rat normal anterior segment by histochemical methods. The distribution of the above hydrolases, particularly that of proteases, varied between ocular tissues and between the three species. Lysosomal hydrolases together with GGT and ALP were consistently active in the corneal epithelium, stroma and endothelium in all three species; the corneal distribution and activity of beta-Gal, APM, APA and DPPIV, however, displayed interspecies variation. The angular tissues showed similarities for most hydrolases with the exceptions of beta-Gal, UE, APM, APA and DPPIV. In all eyes examined strong ciliary epithelial activity for AP, beta-Gal, UE, GGT and ALP was observed in the pars plicata; only the pig eye also displayed strong DPPIV activity in this area. Regional differences in hydrolase distribution in the iris were observed in all species. A post-mortem freezing delay of longer than 24 h resulted in a decrease in hydrolase activity.
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PMID:Hydrolases of anterior segment tissues in the normal human, pig and rat eye: a comparative study. 818 69

A simple and efficient procedure for the construction of secreted fusion proteins in Escherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) of E. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we used lacZ, encoding the cytoplasmic beta-galactosidase (beta-Gal), and phoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that all beta-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in a secA mutant strain only under SecA-deficient conditions.
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PMID:Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins. 884 46


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