A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates

Yasuhiro Hayashi, Yasuhiro Horibata, Keishi Sakaguchi, Nozomu Okino, Makoto Ito

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Glucosylceramide synthase (GlcT) and lactosylceramide synthase (GalT) are key enzymes for the synthesis of major glycosphingolipids of vertebrates. In this article, we report a new reliable method to determine GlcT and GalT activities using the fluorescent acceptor substrates C6-4-nitrobenzo-2-oxa-1,3- diazole (NBD)-ceramide and C6-NBD-glucosylceramide, respectively, and a normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-glucosylceramide for GlcT and C6-NBD-lactosylceramide for GalT, could be separated from the corresponding acceptor substrates within 6 min under the conditions used. Reaction products were able to be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol, making it possible to determine both activities using the lysate from 1 × 10 4 cultured CHOP cells (Chinese hamster ovary cells expressing polyoma LT antigen) and one zebrafish embryo. This method was used successfully to evaluate the degree of knockdown of GlcT and GalT during zebrafish embryogenesis after injection of the morpholino-oligo-based antisense into one- to four-cell embryos. These results indicate that the fluorescence-based HPLC method is a highly sensitive, rapid, and reproducible assay for determining GlcT and GalT activities and is useful for evaluating the activities in gene knockdown experiments.

Original languageEnglish
Pages (from-to)181-186
Number of pages6
JournalAnalytical Biochemistry
Volume345
Issue number2
DOIs
Publication statusPublished - Oct 15 2005

Fingerprint

ceramide glucosyltransferase
High performance liquid chromatography
Assays
High Pressure Liquid Chromatography
Glucosylceramides
Substrates
Zebrafish
Reaction products
Embryonic Structures
Gene Knockdown Techniques
Morpholinos
Glycosphingolipids
Cricetulus
Embryonic Development
Vertebrates
Ovary
Cultured Cells
Genes
Fluorescence
Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates. / Hayashi, Yasuhiro; Horibata, Yasuhiro; Sakaguchi, Keishi; Okino, Nozomu; Ito, Makoto.

In: Analytical Biochemistry, Vol. 345, No. 2, 15.10.2005, p. 181-186.

Research output: Contribution to journalArticle

@article{b41497e4faf1490cb7baa45056031c4f,
title = "A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates",
abstract = "Glucosylceramide synthase (GlcT) and lactosylceramide synthase (GalT) are key enzymes for the synthesis of major glycosphingolipids of vertebrates. In this article, we report a new reliable method to determine GlcT and GalT activities using the fluorescent acceptor substrates C6-4-nitrobenzo-2-oxa-1,3- diazole (NBD)-ceramide and C6-NBD-glucosylceramide, respectively, and a normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-glucosylceramide for GlcT and C6-NBD-lactosylceramide for GalT, could be separated from the corresponding acceptor substrates within 6 min under the conditions used. Reaction products were able to be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol, making it possible to determine both activities using the lysate from 1 × 10 4 cultured CHOP cells (Chinese hamster ovary cells expressing polyoma LT antigen) and one zebrafish embryo. This method was used successfully to evaluate the degree of knockdown of GlcT and GalT during zebrafish embryogenesis after injection of the morpholino-oligo-based antisense into one- to four-cell embryos. These results indicate that the fluorescence-based HPLC method is a highly sensitive, rapid, and reproducible assay for determining GlcT and GalT activities and is useful for evaluating the activities in gene knockdown experiments.",
author = "Yasuhiro Hayashi and Yasuhiro Horibata and Keishi Sakaguchi and Nozomu Okino and Makoto Ito",
year = "2005",
month = "10",
day = "15",
doi = "10.1016/j.ab.2005.05.029",
language = "English",
volume = "345",
pages = "181--186",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates

AU - Hayashi, Yasuhiro

AU - Horibata, Yasuhiro

AU - Sakaguchi, Keishi

AU - Okino, Nozomu

AU - Ito, Makoto

PY - 2005/10/15

Y1 - 2005/10/15

N2 - Glucosylceramide synthase (GlcT) and lactosylceramide synthase (GalT) are key enzymes for the synthesis of major glycosphingolipids of vertebrates. In this article, we report a new reliable method to determine GlcT and GalT activities using the fluorescent acceptor substrates C6-4-nitrobenzo-2-oxa-1,3- diazole (NBD)-ceramide and C6-NBD-glucosylceramide, respectively, and a normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-glucosylceramide for GlcT and C6-NBD-lactosylceramide for GalT, could be separated from the corresponding acceptor substrates within 6 min under the conditions used. Reaction products were able to be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol, making it possible to determine both activities using the lysate from 1 × 10 4 cultured CHOP cells (Chinese hamster ovary cells expressing polyoma LT antigen) and one zebrafish embryo. This method was used successfully to evaluate the degree of knockdown of GlcT and GalT during zebrafish embryogenesis after injection of the morpholino-oligo-based antisense into one- to four-cell embryos. These results indicate that the fluorescence-based HPLC method is a highly sensitive, rapid, and reproducible assay for determining GlcT and GalT activities and is useful for evaluating the activities in gene knockdown experiments.

AB - Glucosylceramide synthase (GlcT) and lactosylceramide synthase (GalT) are key enzymes for the synthesis of major glycosphingolipids of vertebrates. In this article, we report a new reliable method to determine GlcT and GalT activities using the fluorescent acceptor substrates C6-4-nitrobenzo-2-oxa-1,3- diazole (NBD)-ceramide and C6-NBD-glucosylceramide, respectively, and a normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-glucosylceramide for GlcT and C6-NBD-lactosylceramide for GalT, could be separated from the corresponding acceptor substrates within 6 min under the conditions used. Reaction products were able to be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol, making it possible to determine both activities using the lysate from 1 × 10 4 cultured CHOP cells (Chinese hamster ovary cells expressing polyoma LT antigen) and one zebrafish embryo. This method was used successfully to evaluate the degree of knockdown of GlcT and GalT during zebrafish embryogenesis after injection of the morpholino-oligo-based antisense into one- to four-cell embryos. These results indicate that the fluorescence-based HPLC method is a highly sensitive, rapid, and reproducible assay for determining GlcT and GalT activities and is useful for evaluating the activities in gene knockdown experiments.

UR - http://www.scopus.com/inward/record.url?scp=25444458618&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=25444458618&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2005.05.029

DO - 10.1016/j.ab.2005.05.029

M3 - Article

C2 - 16140251

AN - SCOPUS:25444458618

VL - 345

SP - 181

EP - 186

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -