Purification and characterization of plasma membranes obtained from rat neurons prepared by bulk-isolation.
The properties of neuronal plasma membranes are highly specialized, since they include the receptors by which many hormones, neurotransmitters, drugs, and toxins initiate their actions, as well as the components necessary for receiving synaptic input from a variety of sources and for generating the appropriate pattern of action potentials. To be able to study plasma membranes of neurons, it is necessary to separate sufficient quantities of neurons from the other cell types, processes, and myelin found in brain. The methodology for obtaining neurons by bulk-isolation from 10-day-old rat brain has been established, and techniques for purifying the plasma membranes are presented here. The procedure involves swelling the cells in a buffered solution, homogenizing the cells vigorously, and separating the membrane fragments on discontinuous sucrose gradients. The plasma membrane fraction is enriched in plasma membrane marker enzymes and has low activity for contaminating enzymes for subcellular organelles. Electron micrographs show that the fraction consists of membrane vesicles and profiles of various sizes. The protein composition reveals over 60 proteins with molecular weights as high as 100,000 to a low of 14,000. This plasma membrane fraction now can be the focus for studying receptors, glycoproteins, and cell-specific markers of neurons in health and disease.
D015720:5'-Nucleotidase; D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000032:analysis / Q000201:enzymology; D002469:Cell Separation / Q000379:methods*; D008565:Membrane Proteins / Q000302:isolation & purification; D008854:Microscopy, Electron; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D009474:Neurons / Q000032:analysis*; D009708:Nucleotidases / Q000032:analysis; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013347:Subcellular Fractions / Q000032:analysis / Q000201:enzymology / Q000648:ultrastructure
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000254:Sodium-Potassium-Exchanging ATPase