Sterol Analysis in Kluyveromyces lactis
乳酸克鲁维酵母中的甾醇分析
发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2527 浏览次数: 6996
评审: Yanjie LiPierre-Damien DenechaudAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2016
Abstract
Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have been also employed to identify essential steps in the pathway against which antifungals might be developed. We present here a protocol for the isolation of non-saponifiable lipids (sterols) from Kluyveromyces lactis yeast cells and a chromatographic method for quantitative analysis of sterols in lipid extracts (HPLC) that can be performed in laboratories with standard equipment.
Keywords: Ergosterol biosynthesis (麦角甾醇生物合成)
Kluyveromyces lactis (乳酸克鲁维酵母)
Lipid extraction (脂质提取)
High-performance liquid chromatography (高效液相色谱法)
Background
Ergosterol, the primary membrane sterol found in yeast cells, serves a structural role in cellular membranes similar to that of cholesterol in mammalian systems. Sterols have been shown to be responsible for a number of important physical characteristics of membranes by affecting rigidity, fluidity and permeability of membranes. Through their interactions with phospholipids and sphingolipids, sterols are proposed to maintain the lateral heterogeneity of the protein and lipid distribution in the plasma membrane because of their putative role in inducing microdomains called lipid rafts (Dupont et al., 2011; Souza et al., 2011). Sterol biosynthesis in yeast is an energy-expensive, multistep aerobic process, requiring heme and molecular oxygen. Ergosterol and its biosynthetic steps are the major targets for antifungal compounds which have minor effects on cholesterol synthesis of the host organism (Daum et al., 1998). Both ergosterol and some of its biosynthetic intermediates (squalene, 7-dehydrocholesterol) belong to chemicals with a direct positive appeal to people. Therefore a simple and reliable method for sterol isolation is highly rewarding (Valachovic and Hapala, 2017). In this protocol, we describe the analytical method for sterol isolation used for determination of sterol profile in yeast cells.
Materials and Reagents
- Pipette tips (Eppendorf, GBO)
- Inoculation loop
- 15-ml polypropylene centrifuge tubes (Corning, catalog number: 430791 )
- Acid-washed glass beads, diameter 0.45 mm (Sigma-Aldrich, catalog number: G8772 ) (see Note 1)
- Glass Pasteur pipette (Sigma-Aldrich, catalog number: Z628018 )
- 20 ml Pyrex® glass tubes with Teflon cups (Corning) (Sigma-Aldrich, catalog number: Z653527) (see Note 1)
Manufacturer: Pyrex, catalog number: 1622/10M . - Kluyveromyces lactis yeast strain to be analysed
- n-Hexane (anhydrous 95%) (Sigma-Aldrich, catalog number: 439177 ) (see Note 1)
- HPLC standard ergosterol (purity ≥ 95%) (Sigma-Aldrich, catalog number: 45480 ); 0.3 mg/ml stock solution in methanol
- Yeast extract (Biolife Italiana, catalog number: 4122202 )
- Bacto peptone (Biolife Italiana, catalog number: 4122592 )
- D-Glucose (Biolife Italiana, catalog number: 4125012 )
- Potassium hydroxide (KOH) pellets (Merck, catalog number: 1050331000 )
- Methanol (CHROMASOLVTM for HPLC, ≥ 99.9%) (Honeywell, catalog number: 24229 )
- Water (CHROMASOLV® Plus, for HPLC) (Sigma-Aldrich, catalog number: V270733 )
Note: This product has been discontinued. - YEPD rich growth medium (see Recipes)
- Methanolic KOH solution (60% KOH, 50% methanol) (see Note 2 and Recipes)
Equipment
- Pipettes (Eppendorf, HTL)
- Incubation shaker Multitron Standard (Infors HT, Bottmingen, Switzerland)
- Haemocytometer
- Centrifuge 5804R (Eppendorf, model: 5804 R )
- High speed Vortex-Genie 2 (Scientific Industries, model: Digital Vortex-Genie 2 , catalog number: SI-A246)
- Automatic sampler (Shimadzu Scientific Instruments, model: SIL-20AC )
- HPLC instrument (Shimadzu Scientific Instruments, model: Prominence 20A ) equipped with reversed phase C18 column (Ascentis® Express C18, particle size 5 μm, column size 3 x 150 mm, Supelco), UV-Vis detector (Shimadzu Scientific Instruments, model: SPD-20A )
- FastPrep® cell homogenize (MP Biomedicals, model: FastPrep®-24, catalog number: 116004500 )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Gbelska, Y., Toth Hervay, N., Morvova, M. and Konecna, A. (2017). Sterol Analysis in Kluyveromyces lactis. Bio-protocol 7(17): e2527. DOI: 10.21769/BioProtoc.2527.
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分类
微生物学 > 微生物新陈代谢 > 脂质
生物化学 > 脂质 > 脂质测定
生物化学 > 脂质 > 脂质分离
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