Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion-exchange high-performance liquid chromatography with suppressed conductivity detection.
Phosphatidylinositol 4,5-biphosphate (PIP(2)) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP(2) levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP(2) are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP(2) amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 micromol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP(2), whereas overexpression of Type II PI4-kinase can increase both PIP and PIP(2). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and the D3 isomers of PIP(2) are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP(2) remains low; exogenous PIP(2) is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.
Nasuhoglu
Cem
C
Department of Physiology, Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9040, USA.
Feng
Siyi
S
Mao
Janping
J
Yamamoto
Masaya
M
Yin
Helen L
HL
Earnest
Svetlana
S
Barylko
Barbara
B
Albanesi
Joseph P
JP
Hilgemann
Donald W
DW
D019870:1-Phosphatidylinositol 4-Kinase / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D003217:Conductometry / Q000379:methods*; D006168:Guinea Pigs; D006801:Humans; D008563:Membrane Lipids / Q000032:analysis; D009206:Myocardium / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000032:analysis / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000032:analysis / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity
D008563:Membrane Lipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D011994:Recombinant Proteins; D019870:1-Phosphatidylinositol 4-Kinase; D010744:Phosphoric Monoester Hydrolases