放射性 D-erythro-[4,5-³H] 二氢鞘氨醇标记酵母鞘脂的代谢方法

Takefumi  Karashima; Kentaro  Kajiwara; Kouichi Funato

doi:10.21769/BioProtoc.862

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Peer-reviewed

Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-³H]dihydrosphingosine

放射性 D-erythro-[4,5-³H] 二氢鞘氨醇标记酵母鞘脂的代谢方法

TK Takefumi Karashima

KK Kentaro Kajiwara

KF Kouichi Funato email

发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.862 浏览次数: 10016

评审: Fanglian He

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Cover of Molecular Microbiology, featuring study using the protocol.

Dec 2012

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Abstract

Yeast sphingolipids can be metabolically labeled with exogenously added radioactive precursors. Here we describe a method to label ceramides and complex sphingolipids of Saccharomyces cerevisiae with a radioactive ceramide precursor, D-erythro-[4, 5-³H]dihydrosphingosine. This protocol is used to study the biosynthesis, transport and metabolism of sphingolipids in yeast.

Keywords: Sphingolipids (鞘脂)

Yeast (酵母)

Metabolism (代谢)

Radioactive precusor (放射性前体)

Dihydrosphingosine (二氢)

Materials and Reagents

Yeast strain (Saccharomyces cerevisiae)
D-erythro-[4,5-³H]-dihydrosphingosine ([³H]DHS) (60 Ci/mmol) (American Radiolabeled Chemicals, catalog number: ART-46 )
Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S1504 )
Sodium azide (NaN₃) (Sigma-Aldrich, catalog number: S2002 )
Glass beads (0.5 mm) (Yasui Kikai, catalog number: YGB05 )
Chloroform (Sigma-Aldrich, catalog number: 05-3400 )
Methanol (Sigma-Aldrich, catalog number: 19-2410 )
1-Butanol (Sigma-Aldrich, catalog number: 03-4560 )
Ammonium hydroxide (Sigma-Aldrich, catalog number: 221228 )
Acetic acid (Sigma-Aldrich, catalog number: 01-0280 )
Thin layer chromatography silica gel 60 (TLC plate) (aluminium sheet 20 x 20 cm ) (Merck KGaA, catalog number: 105553 )
N₂ gas
Distilled water (H₂O)
Dextrose (D-glucose) (Sigma-Aldrich, catalog number: 07-0680 )
Yeast extract (Oriental Yeast, catalog number: OYC42008000 )
Polypeptone (Wako Chemicals USA, catalog number: 513-76611 )
Yeast nitrogen base without amino acids and ammonium sulfate (BD Difco^TM, catalog number: 233520 )
Adenine (Sigma-Aldrich, catalog number: A9126 )
Leucine (Nacalai Tesque, catalog number: 20327-62 )
Histidine (Nacalai Tesque, catalog number: 18116-92 )
Lysine (Sigma-Aldrich, catalog number: L5626 )
Uracil (Nacalai Tesque, catalog number: 35824-82 )
Ammonium sulfate (Sigma-Aldrich, catalog number: A2939 )
Tryptophan (Nacalai tesque, catalog number: 35607-32 )
Yeast extract-peptone dextrose (YPD) liquid medium (see Recipes)
Synthetic minimal dextrose (SD) liquid medium (see Recipes)
Water-saturated 1-butanol (see Recipes)

Equipment

Tritium-sensitive imaging plate (Fujifilm, catalog number: BAS-TR2040 )
Microcentrifuge tube
Shaking incubator (TAITEC, model: BR-43FL^.MR )
Swinging bucket centrifuge (TOMY, model: LC-201 )
Microcentrifuge (TOMY, model: MX-301 )
Water bath shaker (TAITEC, model: PERSONAL-11 )
Bath-type ultrasonic cleaner (AS ONE, model: US-2R )
Pressure gas blowing concentrator (EYELA, model: MGS-2200 )
TLC developing tank
Hair dryer
FLA-7000 imaging system (GE Healthcare Life Sciences, model: Typhoon FLA 7000 )

Software

FLA-7000 image software (ImageQuant TL analysis)

Procedure

English

中文翻译

文章信息

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如何引用

Karashima, T., Kajiwara, K. and Funato, K. (2013). Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-³H]dihydrosphingosine. Bio-protocol 3(16): e862. DOI: 10.21769/BioProtoc.862.