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)

A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase (2,6ST) has been developed. In the assay an acceptor glycoprotein is immobilized onto microtiter plate wells. The two glycoprotein acceptors used were asialofetuin (ASF), which contains oligosaccharides terminating in the sequence Gal beta 1-4GlcNAc-R, and a neoglycoprotein of bovine serum albumin containing covalently attached Gal beta 1-4GlcNAc-R units. Samples containing the donor CMPNeuAc and the 2,6ST were incubated with the immobilized acceptor to generate the product NeuAc alpha 2-6Gal beta 1-4GlcNAc-R. The product was detected by a biotin-streptavidin system using the biotinylated plant lectin Sambucus nigra agglutinin (SNA), which binds to sialic acid in alpha-2,6, but not in alpha-2,3, linkage. The biotinylated SNA bound to the product was then detected with streptavidin and biotinylated forms of either alkaline phosphatase or the recombinant bioluminescent protein aequorin. The assay was optimized with respect to the commercially available 2,6ST and shown to be dependent on the concentration of acceptor and CMPNeuAc and proportional to the 2,6ST activity in the range of 20 to 400 microU in a 1-h assay. The solid-phase assay also allows for the selective detection of 2,6ST activity in human and fetal bovine serum, where the activity was proportional in the range of 0.1 to 2 microliters of serum.
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PMID:A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase. 128 7

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

We now report a solid-phase assay for CMPNeuAc: Galbeta1-3/4GlcNAc-R alpha-2,3-sialyltransferase (alpha2,3ST) that is nonradioactive and allows specific identification of the sialylated product. An acceptor glycoprotein, desialylated fetuin, is immobilized on a microtiter plate. The transfer of sialic acid from CMPNeuAc generates the product NeuAc alpha2-3Gal beta1-4GlcNAc-R that is specifically bound by biotinylated Maackia amurensis leukoagglutinin (MAL). The binding of biotinylated MAL is measured with either an absorbance-based reagent (streptavidin conjugated to alkaline phosphatase) or a light-based reagent (streptavidin conjugated to the bioluminescent protein aequorin, Aqualite). The rat liver alpha2,3ST was used to optimize the assay. The formation of product is linear with respect to time and dependence on the amounts of CMPNeuAc, enzyme, and acceptor coated on the plates. As little as 0.2 microU of enzyme can be measured using the streptavidin-aequorin reagent. The assay is useful with crude tissue extracts, as demonstrated by the determination of the alpha2,3ST activity in human serum and in microsomes of HL-60 and Chinese hamster ovary cells.
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PMID:Absorbance- and light-based solid-phase assays for CMPNeuAc:Galbeta1-4GlcNAc-R alpha-2,3-sialyltransferase. 861 76

A novel bioluminescence-based solid-phase assay is described for the enzyme GDPFuc:Gal(beta)1-3GlcNAc (Fuc to GlcNAc) alpha1,4-fucosyltransferase (alpha1,4FT), which generates the Lewis a blood group antigen (Le(a)) (Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc-R). Lacto-N-tetraose (LNT,Ga ta)1-3GlcNAc(beta)1-3Gal(beta)1-4Glc) was chemically conjugated to bovine serum albumin (BSA) to generate the acceptor neoglycoprotein LNT-BSA. The Le(a) product of the reaction made in the presence of the donor GDPFuc is detected with a primary monoclonal IgG antibody to Le(a) and a secondary antibody coupled to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Recombinant human GDPFuc:Gal(beta)1-3(4)GlcNAc (Fuc to GlcNAc) alpha1,4/alpha1,3-fucosyltransferase, which exhibits alpha1,4FT activity, was used to optimize the assay. With this assay alpha1,4FT activity is measurable in human serum, in human saliva, and in extracts of the human colon carcinoma cell line SW1116. Activity is absent, however, in extracts of human HL-60 and murine F9 cells, neither of which synthesize Le(a) antigen. Among 10 human donors tested, soluble alpha1,4FT activity, was measurable in serum and saliva of some, but not all donors. However, the presence of enzyme activity in sera does not correlate with Lewis blood group phenotype of erythrocytes. The saliva of one donor, which contained Le(a) antigens, exhibited no alpha1,4FT activity. That saliva was found to contain a heat-stable factor(s) capable of inhibiting the alpha1,4FT activity when mixed with donor saliva containing alpha1,4FT activity. This new assay should be useful in assessing the Lewis enzyme activity in body fluids and its relationship to the Lewis blood group status on cells and secreted glycoconjugates in normal and diseased states.
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PMID:alpha1,4-Fucosyltransferase activity in human serum and saliva. 891 40

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity. A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation. The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures. Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.
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PMID:Secretion of alpha1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera. 951 86

We have developed a dual-analyte chemiluminescence hybridization assay for quantitative polymerase chain reaction (PCR). The method allows simultaneous determination of both amplified target DNA and internal standard (IS) in the same reaction vessel. The target DNA from the sample (233 bp) was coamplified with a constant amount of a recombinant DNA IS that had the same size and primer binding regions as the target DNA, differing only by a 24-bp sequence, centrally located. Biotinylated PCR products from target DNA and IS were captured on a single microtiter well coated with streptavidin. The amplified target DNA was hybridized with a digoxigenin-labeled specific probe, and the hybrids were determined by using antidigoxigenin antibody labeled with aequorin. The amplified DNA IS was hybridized, in the same well, with a fluorescein-labeled probe, and the hybrids were determined by using an antifluorescein antibody conjugated to alkaline phosphatase. Aequorin was measured by adding a Ca(2+)-containing light-triggering solution. Alkaline phosphatase was measured by using a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained from the target DNA and IS amplification products was linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear range extended from 430 to 315,000 target DNA molecules. Average CVs ranged from 7 to 17%. The proposed system is expected to facilitate the automation and routine use of quantitative PCR.
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PMID:Quantitative polymerase chain reaction based on a dual-analyte chemiluminescence hybridization assay for target DNA and internal standard. 978 50

The difference in light-emission kinetics between the Ca(2+)-triggered bioluminescent reaction of the photoprotein aequorin (AEQ) and the alkaline phosphatase (ALP)-catalyzed chemiluminescent hydrolysis of dioxetane aryl phosphate substrates was exploited for the analysis of both alleles of biallelic polymorphisms in a single microtiter well. The genotyping of the IVS-1-110 locus of the human beta-globin gene was chosen as a model. Genomic DNA, isolated from whole blood, was first subjected to polymerase chain reaction using primers flanking the polymorphic site. A single oligonucleotide-ligation reaction employing two allele-specific probes, labeled with biotin and digoxigenin, and a common probe carrying a characteristic tail was then performed. The ligation products were captured in a microtiter well through hybridization of the tail with an immobilized complementary oligonucleotide. The products were detected by adding a mixture of streptavidin-aequorin complex and antidigoxigenin-alkaline phosphatase conjugate. AEQ was measured first by adding Ca(2+) and integrating the signal for 3s followed by the addition of the substrate for ALP. The ratio of the luminescence signals obtained from ALP and AEQ gives the genotype of each sample. The coefficient of variation of the dual assay ranged from 7 to 11% for each allele. The reproducibility of the ALP/AEQ signal ratio was about 14%. The proposed assay allows for many samples to be screened in parallel in a single microtiter plate, for single-nucleotide polymorphisms.
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PMID:Combined flash- and glow-type chemiluminescent reactions for high-throughput genotyping of biallelic polymorphisms. 1292 33

Primer extension reaction (PEXT) is the most widely used approach to genotyping of single nucleotide polymorphisms (SNP). It is based on the high accuracy of nucleotide incorporation by the DNA polymerase. We propose a dual-analyte bio/chemiluminometric method for the simultaneous detection of the PEXT reaction products of the normal and mutant allele in a high sample-throughput format. PCR-amplified DNA fragments that span the SNP of interest are subjected to two PEXT reactions using normal and mutant primers in the presence of digoxigenin-dUTP and biotin-dUTP. Both primers contain a d(A)30 segment at the 5'-end but differ in the final nucleotide at the 3'-end. Under optimized conditions only the primer that is perfectly complementary with the interrogated DNA will be extended by DNA polymerase and lead to a digoxigenin- or biotin-labeled product. The products of the PEXT reactions are mixed, denatured, and captured in microtiter wells through hybridization with immobilized oligo(dT) strands. Detection is performed by adding a mixture of antidigoxigenin-alkaline phosphatase (ALP) conjugate and a streptavidin-aequorin conjugate. The flash-type bioluminescent reaction of aequorin is triggered by the addition of Ca2+. ALP is then measured by adding the appropriate chemiluminogenic substrate. The method was evaluated by genotyping two SNPs of the human mannose-binding lectin gene (MBL2) and one SNP of the cytochrome P450 gene CYP2D6. Patient genotypes showed 100% concordance with direct DNA sequencing data.
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PMID:Genotyping of single nucleotide polymorphisms by primer extension reaction and a dual-analyte bio/chemiluminometric assay. 1755 27

We developed a highly sensitive quadruple-analyte chemiluminometric hybridization assay for simultaneous quantification of four nucleic acid sequences. The targets are amplified by the polymerase chain reaction (PCR) and captured to microtiter wells coated with streptavidin. The immobilized fragments are hybridized with specific probes containing a sequence complementary to the target and a sequence or a hapten that allows linkage with a chemiluminescent reporter. We prepared a mixture of four reporters conjugated to complementary oligonucleotides or antihapten antibodies. The reporters were aequorin-(dT)(30), galactosidase-oligonucleotide, horseradish peroxidase-antifluorescein, and alkaline phosphatase-antidigoxigenin conjugates. The four chemiluminescent reactions were triggered sequentially. The signals were linearly related to the concentration of target sequences. The entire quadruple-analyte bioluminometric hybridization assay is complete in 75 min. We have demonstrated the applicability of the proposed assay to high-throughput quantitative competitive PCR of two target sequences in the presence of the corresponding competitors. The assay is universal since the same reporter conjugates can be used for multianalyte quantification of any sequences with properly designed probes.
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PMID:Quadruple-analyte chemiluminometric hybridization assay. Application to double quantitative competitive polymerase chain reaction. 1799 78

We compare aequorin, alkaline phosphatase and horseradish peroxidase as reporters for luminescent immunoassays using alpha-fetoprotein (AFP) as a model analyte. Biotinylated aequorin was prepared from mutated aequorin containing a reactive cysteine residue by chemical conjugation. The measurable range of AFP using this biotinylated aequorin was 0.02-200 ng/ml, with a lower background level than the other biotinylated enzymes tested.
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PMID:Comparison of luminescent immunoassays using biotinylated proteins of aequorin, alkaline phosphatase and horseradish peroxidase as reporters. 1906 Mar 86


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