Isolation of postsynaptic densities retaining their membrane attachment.
A method is described for the preparation of a subcellular fraction, 30-50% pure, of intact postsynaptic units from rat cerebral cortex. The isolation procedure is based on chemical dissociation of the synaptic cleft as described by Crawford, Osborne & Potter followed by sonication of the extracted membranes and separation of the postsynaptic units on a discontinuous sucrose gradient. This preparation provides the first practical procedure for the isolation of postsynaptic densities, prominent organelles of unknown function, without the use of detergents, enabling retention of the postsynaptic membrane in association with the postsynaptic density. The preparation shows enhanced binding of spiroperidol, a dopamine agonist, which, in conjunction with morphological evidence, indicates that the preparation is sufficiently intact to enable study of the interaction of the postsynaptic membrane with the postsynaptic density. Actin, alpha- and beta-tubulin and postsynaptic density protein constitute the major proteins in the preparation; they are present in amounts of 41, 54, 57 and 74 micrograms per mg protein, respectively; as compared to 54, 59, 55 and 9 micrograms per mg protein of the synaptic junctional membrane used as starting material. The utility of the preparation for a number of localization studies, including ion translocating adenosine 5'-triphosphatases, protein kinases and their substrates is discussed.
D000199:Actins / Q000378:metabolism; D000818:Animals; D001369:Axons / Q000648:ultrastructure*; D002458:Cell Fractionation; D002469:Cell Separation / Q000379:methods*; D002540:Cerebral Cortex / Q000166:cytology*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D016900:Neurofilament Proteins; D051381:Rats; D013134:Spiperone / Q000378:metabolism; D013569:Synapses / Q000648:ultrastructure*; D013570:Synaptic Membranes / Q000648:ultrastructure*; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D014404:Tubulin; D013134:Spiperone