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 part icles in conjunction with high gradient magnetic affinity chromatograp hy (HIMAC) to prepare plasma membranes from cultured cells. Membrane-i mpermeable succinimidyl esters inactivate alkaline phosphodiesterase 1 (APDE-1) and were used to establish the proportion of APDE-1 expresse d at the cell surface. The yield of inhibitable APDE-1 provides an acc urate indication of plasma membrane yield, which was >90% for Chinese hamster ovary (CHO) cells. Plasma membranes prepared by HIMAC containe d <5-13% of endoplasmic reticulum, Golgi, mitochondria, lysosomes, or endosomes. Pulse-chase experiments performed with the alpha5beta1 inte grin receptor confirmed the high yield of plasma membrane and demonstr ated the utility of this procedure for examining trafficking of protei ns 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 chole sterol. Phosphatidylserine was the only glycerophospholipid highly enr iched (71%) in the retained fraction. The glycosphingolipids lactosylc eramide and ganglioside G(M3) were enriched in the plasma membrane fra ction to the same extent as sphingomyelin. The major fraction of the g lycosphingolipid precursors glucosylceramide and ceramide was localize d to intracellular membranes. These findings indicate that the plasma membrane of CHO cells contains approximately half of the total cellula r phospholipids and an even higher percentage of sphingomyelin and cho lesterol. The high efficiency and rapidity of this isolation procedure should aid the analysis of plasma membrane components significantly.