Extraction and analysis of soluble inositol polyphosphates from yeast

被引：91
作者：
Azevedo, Cristina
Saiardi, Adolfo
机构：
[1] UCL, MRC, Cell Biol Unit, London WC1E 6BT, England
[2] UCL, Dept Biochem & Mol Biol, Mol Cell Biol Lab, London WC1E 6BT, England
来源：
NATURE PROTOCOLS | 2006年 / 1卷 / 05期
基金：
英国医学研究理事会;
关键词：
D O I：
10.1038/nprot.2006.337
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
Soluble inositol polyphosphates are implicated in the regulation of many important cellular functions. This protocol to extract and separate inositol polyphosphates from Saccharomyces cerevisiae is divided into three steps: labeling of yeast, extraction of soluble inositol polyphosphates and chromatographic separation. Yeast cells are incubated with tritiated inositol, which is taken up and metabolized into different phosphorylated forms. Soluble inositol polyphosphates are then acid-extracted and fractionated by high-performance liquid chromatography. The radioactivity of each fraction is determined by scintillation counting. This highly sensitive and reproducible method allows the accurate detection of subtle changes in the inositol polyphosphate profile and takes less than 48 h. It can easily be applied to other systems and we have included two adaptations of the protocol, one optimized for mammalian cells and the other for Arabidopsis thaliana.
引用
收藏
页码：2416 / 2422
页数：7
相关论文
共 50 条
[31]   Membrane-permeant bioactivatable inositol polyphosphates [J].
Schultz, C ;
Rudolf, MT ;
Gillandt, H ;
TraynorKaplan, AE .
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 1997, 214 :65-CARB
[32]   Synthesis of Unsymmetric Diphospho-Inositol Polyphosphates [J].
Capolicchio, Samanta ;
Thakor, Divyeshsinh T. ;
Linden, Anthony ;
Jessen, Henning J. .
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2013, 52 (27) :6912-6916
[33]   Role of inositol polyphosphates in programmed cell death [J].
Agarwal, Rakhee ;
Mumtaz, Hamid ;
Ali, Nawab .
MOLECULAR AND CELLULAR BIOCHEMISTRY, 2009, 328 (1-2) :155-165
[34]   Role of inositol polyphosphates in programmed cell death [J].
Rakhee Agarwal ;
Hamid Mumtaz ;
Nawab Ali .
Molecular and Cellular Biochemistry, 2009, 328 :155-165
[35]   Metabolism of inositol 1,4,5-trisphosphate in Candida albicans: Significance as a precursor of inositol polyphosphates and in signal transduction during the dimorphic transition from yeast cells to germ tubes [J].
Gadd, GM ;
Foster, SA .
MICROBIOLOGY-SGM, 1997, 143 :437-448
[36]   ISOLATION AND CHARACTERIZATION OF POLYPHOSPHATES FROM THE YEAST-CELL SURFACE [J].
TIJSSEN, JPF ;
DUBBELMAN, TMAR ;
VANSTEVENINCK, J .
BIOCHIMICA ET BIOPHYSICA ACTA, 1983, 760 (01) :143-148
[37]   New Horizons in Cellular Regulation by Inositol Polyphosphates: Insights from the Pancreatic β-Cell [J].
Barker, Christopher J. ;
Berggren, Per-Olof .
PHARMACOLOGICAL REVIEWS, 2013, 65 (02) :641-669
[38]   COMPLETE CHARACTERIZATION OF MYO-INOSITOL POLYPHOSPHATES FROM BEEF BRAIN PHOSPHOINOSITIDE [J].
TOMLINSON, R ;
BALLOU, CE .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1961, 236 (07) :1902-&
[39]   ACCUMULATION OF [3H]-INOSITOL INTO INOSITOL POLYPHOSPHATES DURING DEVELOPMENT OF DICTYOSTELIUM [J].
EUROPEFINNER, GN ;
GAMMON, B ;
NEWELL, PC .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1991, 181 (01) :191-196
[40]   GEL CHROMATOGRAPHY OF INOSITOL POLYPHOSPHATES AND AVIAN HAEMOGLOBIN-INOSITOL PENTAPHOSPHATE COMPLEX [J].
STEWARD, JH ;
TATE, ME .
JOURNAL OF CHROMATOGRAPHY, 1969, 45 (3-4) :400-&
12345