Characterization of plasma membranes from A431 cells, isolated by self-generating Percoll gradient: a rapid isolation procedure to obtain plasma membranes with functional epidermal growth factor receptors.
Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers. Plasma membranes displayed a 4.5- and 4.4-fold enrichment in [3H]concanavalin A and 5'-nucleotidase, respectively. This proteic fraction was further characterized by its lipid composition and phospholipid analysis. The cholesterol/phospholipid molar ratio was 0.45 in plasma membranes against 0.19 in lysate. Sphingomyelin increased from 7.5% of total phospholipids in lysate to 16.2% in plasma membranes, as well as phosphatidylserine which displayed a 1.5-fold enrichment in the plasma membrane fraction. This was at the expense of phosphatidylcholine (45.2% in lysate, against 35% in plasma membranes). Electron microscopy of the isolated material showed vesicles essentially free from endoplasmic reticulum and organelles. These plasma membranes retained the ability to bind 125I-labelled epidermal growth factor (125I-EGF) with a Kd = 4.7 nM and Bmax = 63 pmol/mg protein. EGF binding resulted in a stimulation of the phosphorylation protein reaction in the presence of [gamma-32P]ATP and sodium dodecyl sulfate polyacrylamide gels of phosphorylated proteins indicated that the radioactivity of the major band of molecular weight 170,000 was clearly enhanced by EGF binding. These results indicate that the EGF receptor and its intrinsic protein kinase activity were preserved during our plasma membrane isolation procedure.
Payrastre
B
B
INSERM Unité 101, Hôpital Purpan, Toulouse, France.
Plantavid
M
M
Etievan
C
C
Ribbes
G
G
Carratero
C
C
Chap
H
H
Douste-Blazy
L
L
D002294:Carcinoma, Squamous Cell / Q000648:ultrastructure*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000032:analysis; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008563:Membrane Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D010713:Phosphatidylcholines / Q000032:analysis; D010716:Phosphatidylinositols / Q000032:analysis; D010718:Phosphatidylserines / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013109:Sphingomyelins / Q000032:analysis; D014407:Tumor Cells, Cultured
D008563:Membrane Lipids; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010743:Phospholipids; D013109:Sphingomyelins; D004815:Epidermal Growth Factor; D002784:Cholesterol; D011494:Protein Kinases; D066246:ErbB Receptors