Abstract
Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% Na 2 B 4 O 7 . Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer β1-4-galactosyltransferase), (Km of glucosylceramide = 1.65 × 10 −4 M; Km for UDP-Gal = 0.5 × 10 −4 M; V max is determined in the presence of optimum detergent concentrations (2–15 mg/ml of Cutscum–Triton X-100, 2:1); Mn ++ and Mg ++ , 10–20 mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.
| Original language | English (US) |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press Inc |
| Pages | 189-205 |
| Number of pages | 17 |
| DOIs | |
| State | Published - Jan 1 2018 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 1804 |
| ISSN (Print) | 1064-3745 |
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Keywords
- GalT-2
- Glycosyltransferases
- Lactosylceramide synthase
ASJC Scopus subject areas
- Molecular Biology
- Genetics
Cite this
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Protocols for glycosyltransferase assays : Ganglioside globoside and lewis-X intermediate-lactosylceramide biosyntheses in eukaryotic systems. / Basu, Subhash; Agarwal, Arun; Basu, Manju; Ma, Rui; Moskal, Joseph R.
Methods in Molecular Biology. Humana Press Inc, 2018. p. 189-205 (Methods in Molecular Biology; Vol. 1804).Research output: Chapter in Book/Report/Conference proceeding › Chapter
TY - CHAP
T1 - Protocols for glycosyltransferase assays
T2 - Ganglioside globoside and lewis-X intermediate-lactosylceramide biosyntheses in eukaryotic systems
AU - Basu, Subhash
AU - Agarwal, Arun
AU - Basu, Manju
AU - Ma, Rui
AU - Moskal, Joseph R
PY - 2018/1/1
Y1 - 2018/1/1
N2 - Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% Na 2 B 4 O 7 . Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer β1-4-galactosyltransferase), (Km of glucosylceramide = 1.65 × 10 −4 M; Km for UDP-Gal = 0.5 × 10 −4 M; V max is determined in the presence of optimum detergent concentrations (2–15 mg/ml of Cutscum–Triton X-100, 2:1); Mn ++ and Mg ++ , 10–20 mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.
AB - Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% Na 2 B 4 O 7 . Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer β1-4-galactosyltransferase), (Km of glucosylceramide = 1.65 × 10 −4 M; Km for UDP-Gal = 0.5 × 10 −4 M; V max is determined in the presence of optimum detergent concentrations (2–15 mg/ml of Cutscum–Triton X-100, 2:1); Mn ++ and Mg ++ , 10–20 mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.
KW - GalT-2
KW - Glycosyltransferases
KW - Lactosylceramide synthase
UR - http://www.scopus.com/inward/record.url?scp=85048953732&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85048953732&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-8552-4_8
DO - 10.1007/978-1-4939-8552-4_8
M3 - Chapter
T3 - Methods in Molecular Biology
SP - 189
EP - 205
BT - Methods in Molecular Biology
PB - Humana Press Inc
ER -