The structure of postsynaptic densities isolated from dog cerebral cortex: II. characterization and arrangement of some of the major proteins within the structure

Abstract

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein was tentatively labeled neurofilament protein, on the basis of its 51,000 MW correspondence to a protein found in neurofilament preparations. Other proteins were akin to some dog myofibrillar proteins, on the basis of immunological cross-reaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin was not present in the PSD fraction, a major protein present was actin, as evident from reactivity with antiactin serum, from SDS-gel mobility and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted to extract some proteins, as revealed by gel electrophoresis, and to observe the structual changes of the PSD fraction residue after extraction of these proteins. The PSD was remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 MW protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca2+ extractor, ethylene glycol-bis (.beta.-aminoethyl ether) N,N,N'',N''-tetraacetate (EGTA) and guanidine caused an opening up of the native dense PSD structure, revealing .apprx. 10 nm filaments, presumably consisting of neurofilament protein. Both DTT-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the EM, revealed both 10 nm and 3-5 nm filaments; the latter could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 MW component and 2 minor proteins of 68,000 and 73,00 MW. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10 nm and 3-5 nm filaments, held together through Ca2+ interaction and by bonds amendable to breakage by sulfhydryl-blocking and disulfide-reducing reagents; either removal of Ca2+ and/or rupture of these disulfide bonds opens up the structure. On the basis of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory was presented which envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its proteins relative to one another.