Isolation of plasma membranes from neurons grown in primary culture.
Plasma membranes from chick embryo neuronal primary cultures were isolated after subjecting 5-day-old cells, previously surface labeled with either lactoperoxidase-catalyzed radioiodination or galactose oxidase/NaB3H4, to a freeze-thaw cycle. The cellular material adhering to the culture substratum was washed, and the "wash" fractions were pooled and centrifuged at 37,000g. The resulting pellet was resuspended in 3 ml of buffer, layered on 33 ml of 33% sucrose, and centrifuged at 105,000g. Radioactivity was recovered at the top of the gradient. Sedimentation of these fractions and biochemical studies revealed that the pellet was 20- and 12-fold enriched in (Na+,K+)-adenosinetriphosphatase and 5'-nucleotidase, respectively. The preparation was devoid of inner mitochondrial (succinate dehydrogenase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), and Golgi (UDP galactose:N-acetylglucosamine galactosyltransferase) enzymatic markers. Ultrastructural studies showed that the membrane preparation was homogeneous and lacked mitochondria endoplasmic reticulum and lysosomes. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of 11 protein components with molecular masses ranging from 120 to 300 kDa. This method for the isolation of plasma membranes probably depends on the capacity of the cellular material to adhere to the culture substratum and to entrap intracellular organelles during the freeze-thaw cycle. The membrane preparation seems suitable for studying the function of high-molecular-weight protein components of neuronal plasma membranes.
Mersel
M
M
Centre de Neurochimie du CNRS, Strasbourg, France.
Lelong
I
I
Hindelang
C
C
Sarlieve
L
L
Vincendon
G
G
D015720:5'-Nucleotidase; D000818:Animals; D002462:Cell Membrane / Q000032:analysis* / Q000201:enzymology / Q000648:ultrastructure; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000032:analysis; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D008995:Monoamine Oxidase / Q000378:metabolism; D009474:Neurons / Q000032:analysis* / Q000201:enzymology / Q000648:ultrastructure; D009708:Nucleotidases / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism
D005954:Glucosephosphate Dehydrogenase; D013385:Succinate Dehydrogenase; D008995:Monoamine Oxidase; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000254:Sodium-Potassium-Exchanging ATPase; D005690:Galactose