Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradient magnetic affinity chromatography.
We have utilized wheat germ agglutinin conjugated to iron/dextran particles in conjunction with high gradient magnetic affinity chromatography (HIMAC) to prepare plasma membranes from cultured cells. Membrane-impermeable succinimidyl esters inactivate alkaline phosphodiesterase 1 (APDE-1) and were used to establish the proportion of APDE-1 expressed at the cell surface. The yield of inhibitable APDE-1 provides an accurate indication of plasma membrane yield, which was > 90% for Chinese hamster ovary (CHO) cells. Plasma membranes prepared by HIMAC contained < 5-13% of endoplasmic reticulum, Golgi, mitochondria, lysosomes, or endosomes. Pulse-chase experiments performed with the alpha 5 beta 1 integrin receptor confirmed the high yield of plasma membrane and demonstrated the utility of this procedure for examining trafficking of proteins to and from the plasma membrane. We determined the lipid content of plasma membranes prepared by HIMAC. CHO plasma membranes contain 49% of total cellular phospholipid, 69% of sphingomyelin, and 64% of cholesterol. Phosphatidylserine was the only glycerophospholipid highly enriched (71%) in the retained fraction. The glycosphingolipids lactosylceramide and ganglioside GM3 were enriched in the plasma membrane fraction to the same extent as sphingomyelin. The major fraction of the glycosphingolipid precursors glucosylceramide and ceramide was localized to intracellular membranes. These findings indicate that the plasma membrane of CHO cells contains approximately half of the total cellular phospholipids and an even higher percentage of sphingomyelin and cholesterol. The high efficiency and rapidity of this isolation procedure should aid the analysis of plasma membrane components significantly.
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
D E
DE
Warnock
C
C
Roberts
M S
MS
Lutz
W A
WA
Blackburn
W W
WW
Young
J U
JU
Baenziger
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016023:Integrins / Q000032:analysis / Q000096:biosynthesis; D007700:Kinetics; D008280:Magnetics; D008563:Membrane Lipids / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D016023:Integrins; D008563:Membrane Lipids; D008565:Membrane Proteins; D011994:Recombinant Proteins; D013388:Succinimides; C081067:sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I