Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.13.12.5 (aequorin)
 1,451 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.
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PMID:A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin. 190 13

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

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