GLA activity was measured as described previously with minor modification59 (link). Three μg of protein in 20 μL of lysis buffer (3 mg/mL sodium taurocholate, 28 mM citric acid, and 44 mM Na2HPO4) was added to 80 μL of assay buffer (5 mM p-nitrophenyl α-D- galactopyranoside, 28 mM citric acid, 44 mM Na2HPO4, and 5 mg/mL BSA with 117 mM N-acetyl-D-galactosamine) in a 96-well plate. GLA activity in the cell lysates were calculated using α-galactosidase (Sigma) as reference, which hydrolyzes 1.0 μmol of substrate to p-nitrophenol and D-galactose per minute. Incubations proceeded at 37°C for 1 hr. Stop solution (100 μL of 200 mM Na2CO3) was added, and fluorescence was read on a plate reader (SpectraMax 250, Molecular devices) at 400 nm.
α galactosidase
Manufactured by Merck Group
Sourced in United States
α-galactosidase is an enzyme that catalyzes the hydrolysis of terminal, non-reducing α-D-galactose residues in α-D-galactosides. It is used in various laboratory applications, including the analysis of glycoprotein structures and the detection of genetic disorders.
Automatically generated - may contain errors
4 protocols using α galactosidase
1
Assay for α-Galactosidase Activity
2
Structural Analysis of Glycolipids
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The structures of the further purified glycolipids were also determined by negative ion fast atom bombardment mass spectrometry (FABMS) and glycosidase treatment. Approximately 5 µg of an isolated neutral glycolipid in 5 µl of chloroform/methanol (1:1, v/v) was mixed with ~5 µl of triethanolamine, and the resultant solution was placed on a stainless steel sample holder for FABMS. Analysis was performed by bombardment with a neutral xenon beam with a kinetic energy of 4 keV, and detection of negative ions was performed with a mass spectrometer (JMSHX-110; JEOL, Ltd., Tokyo, Japan) equipped with a JMA-5500 computer system (JEOL, Ltd.). Assignment of mass numbers was achieved by comparing the spectrum with that of perfluoroalkyl phosphazine (Ultramark 1621; PCR, Inc., Gainesville, FL, USA).
TLC upon treatment with α-galactosidase (Sigma-Aldrich) was performed to confirm the sequence of the sugar chains.
TLC upon treatment with α-galactosidase (Sigma-Aldrich) was performed to confirm the sequence of the sugar chains.
3
Enzymatic Characterization of α-Galactosidase
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α-Galactosidase from green coffee bean (pH optimum 5.3 and no data for temperature optimum) was purchased from Sigma-Aldrich. To a suspension of the enzyme (ca. 25 U) in 3.2 M ammonium sulfate (pH 6.0) (100 μL) was added 1.0 mL of phosphate-citrate buffer prepared at pH 5.3, giving the enzyme solution. The synthetic X-α-Gal (1.0 mg) was added to the enzyme solution (275 μL) and the enzyme activity at 40 °C was monitored in 10 min, 30 min, and 2 h.
4
Exoglycosidase Profiling of R27T Glycans
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The 2-AB-labeled glycans derived from R27T were incubated overnight
at 37 °C with various exoglycosidase enzymes in 50 mM sodium
acetate of pH 5.5. The following exoglycosidases were used: sialidase
(QABio, USA) from Arthrobacter ureafaciens (a368S); specific for α2-3, -6, -8, and -9 sialic acids; beta-galactosidase
(QABio) from Streptococcus pneumoniae (b4G; specific for β1-4 galactose); α-galactosidase
(Sigma, USA) from green coffee bean (a36G; specific for α1-3/6
galactose); fucosidase (QABio) from Almond meal (a34F; specific for
α1-3 and -4 fucose); fucosidase (Sigma, USA) from Bovine kidney
(a6F; specific for α1-6 > 2 fucose); and N-acetylglucosaminidase
(QABio) from S. pneumoniae (sph; specific
for β-GlcNAc). Enzymes were removed by a protein-binding membrane,
and samples were analyzed by HILIC–HPLC using an LSN2-40m-Nlink-35%_G100
and a LudgerSep-N2 column (LS-N24.6 × 150) on a Waters 2795 HPLC
instrument linked to a 2475 fluorescence detector controlled by Empower
software version 2, build 2154. Buffer A was 50 mM ammonium formate
made from Ludger stock buffer # LS-BUFAMMFORM-2M-50ML, and Buffer
B was acetonitrile (190 far-UV/gradient quality; Romil #H049). Samples
were injected in 35% aqueous buffer/65% acetonitrile (35 μL
samples +65 μL of acetonitrile), and the injection volume was
25 μL.
at 37 °C with various exoglycosidase enzymes in 50 mM sodium
acetate of pH 5.5. The following exoglycosidases were used: sialidase
(QABio, USA) from Arthrobacter ureafaciens (a368S); specific for α2-3, -6, -8, and -9 sialic acids; beta-galactosidase
(QABio) from Streptococcus pneumoniae (b4G; specific for β1-4 galactose); α-galactosidase
(Sigma, USA) from green coffee bean (a36G; specific for α1-3/6
galactose); fucosidase (QABio) from Almond meal (a34F; specific for
α1-3 and -4 fucose); fucosidase (Sigma, USA) from Bovine kidney
(a6F; specific for α1-6 > 2 fucose); and N-acetylglucosaminidase
(QABio) from S. pneumoniae (sph; specific
for β-GlcNAc). Enzymes were removed by a protein-binding membrane,
and samples were analyzed by HILIC–HPLC using an LSN2-40m-Nlink-35%_G100
and a LudgerSep-N2 column (LS-N24.6 × 150) on a Waters 2795 HPLC
instrument linked to a 2475 fluorescence detector controlled by Empower
software version 2, build 2154. Buffer A was 50 mM ammonium formate
made from Ludger stock buffer # LS-BUFAMMFORM-2M-50ML, and Buffer
B was acetonitrile (190 far-UV/gradient quality; Romil #H049). Samples
were injected in 35% aqueous buffer/65% acetonitrile (35 μL
samples +65 μL of acetonitrile), and the injection volume was
25 μL.
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