Postsynaptic densities (PSDs) have been isolated from cerebral cortex, midbrain, cerebellum, and brain stem by the Triton X-100 method previously used in the isolation of cerebral PSDs (Cohen et al ., 1977, 1. Cell Biol. 74 :181) . These PSDs have been compared in protein composition, protein phosphorylation, and morphology . Thin-section electron microscopy revealed that cerebral cortex and midbrain PSDs were identical, being^-57 nm thick and composed of apparent aggregates 20-30 nm in diameter . Isolated cerebellar PSDs appeared thinner (33 nm) than cerebral cortex PSDs and lacked the apparent 20-to 30-nm aggregates, but had a latticelike structure. In unidirectional and rotary-shadowed replicas, the cerebrum and midbrain PSDs were circular in shape with a large central perforation or hole in the center of them . Cerebellum PSDs did not have a large perforation, but did have numerous smaller perforations in a lattice like structure. Filaments (6-9 nm) were observed connecting possible 20-to 30-nm aggregates in cerebrum PSDs and were also observed radiating from one side of the PSD. Both cerebral cortex and midbrain PSDs exhibited identical protein patterns on SDS gel electrophoresis. In comparison, cerebellar PSDs (a) lacked the major 51,000 Mr protein, (b) contained two times less calmodulin, and (c) contained a unique protein at 73,000 Mr. Calcium plus calmodulin stimulated the phosphorylation of the 51,000 and 62,000 Mr bands in both cerebral cortex and midbrain PSDs . In cerebellar PSDs, only the 58,000 and 62,000 M r bands were phosphorylated . In the PSDs from all brain regions, CAMP stimulated the phosphorylation of Protein la (73,000 Mr), Protein Ib (68,000 Mr), and a 60,000 Mr protein, although cerebrum and midbrain PSDs contained very much higher levels of phosphorylated protein than did the cerebellum .On the basis of the morphological criteria, it is possible that PSDs isolated from cerebrum and midbrain were derived from the Gray type I, or asymmetric, synapses, whereas cerebellum PSDs were derived from the Gray type II, or symmetric, synapses, Since there is some evidence that the type I synapses are involved in excitatory mechanisms while he type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed .

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of the methods of Davis and Bloom (21,22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resolution SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nucleic acids, and very little phospholipid being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 % that of a mitochondrial fraction) and a small amount of 5'-nucleotidase activity (of a specific activity between 6 and 7% that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures -400 nm long and -40 nm wide, made up of apparent particles 13-28 nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter -400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing 125I-labeled myelin, synaptic vesicle, and mitochondrial fraction protel'~S with synaptosomes, and then isolating the density

Rat liver, liver homogenates, and microsome fractions separated therefrom were examined systematically in the electron microscope in sections of OsO4-fixed, methacrylate-embedded tissue and pellets. It was found that most microsomes are morphologically identical with the rough surfaced elements of the endoplasmic reticula of hepatic cells. They appear as isolated, membrane-bound vesicles, tubules, and cisternae which contain an apparently homogeneous material of noticeable density, and bear small, dense particles (100 to 150 A) attached to their outer aspect. In solutions of various osmolar concentrations they behave like osmometers. The findings suggest that they derive from the endoplasmic reticulum by a generalized pinching-off process rather than by mechanical fragmentation. The microsome fractions contain in addition relatively few vesicles free of attached particles, probably derived from the smooth surfaced parts of the endoplasmic reticula. Dense, peribiliary bodies represent a minor component of the same fractions. The microsomes derived from 1 gm. wet weight liver pulp contained (averages of 10 experiments) 3.09 mg. protein N, 3.46 mg. RNA (RNA/protein N = 1.12), and 487 µg. phospholipide P. They displayed DPNH-cytochrome c reductase activity and contained an alcohol-soluble hemochromogen. The microsome preparations proved resistant to washing and "aging." Treatment with versene and incubation with ribonuclease (30 minutes at 37°C.) resulted in appreciable losses of RNA and in partial or total disappearance of attached particles. Treatment with deoxycholate (0.3 to 0.5 per cent, pH = 7.5) induced a partial clarification of the microsome suspensions which, upon centrifugation, yielded a small pellet of conglomerated small, dense particles (100 to 150 A) with only occasionally interspersed vesicles. The pellet contained ∼80 to 90 per cent of the RNA and ∼20 per cent of the protein N of the original microsomes. The supernatant accounted satisfactorily for the materials lost during deoxycholate treatment. The findings suggest that the microsomal RNA is associated with the small particles whereas most of the protein and nearly all of the phospholipide, hemochromogen, and DPNH-cytochrome c reductase activity are associated with the membrane or content of the microsomes.

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Subfractionation of preparations of rat liver microsomes with a suitable concentration of sodium deoxycholate has resulted in the isolation of a membrane fraction consisting of smooth surfaced vesicles virtually free of ribonucleoprotein particles. The membrane fraction is rich in phospholipids, and contains the microsomal NADH-cytochrome c reductase, NADH diaphorase, glucose-6-phosphatase, and ATPase in a concentrated form. The NADPH-cytochrome c reductase, a NADPH (or pyridine nucleotide unspecific) diaphorase, and cytochrome b5 are recovered in the clear supernatant fraction. The ribonucleoprotein particles are devoid of, or relatively poor in, the enzyme activities mentioned. Those enzymes which are bound to the membranes vary in activity according to the structural state of the microsomes, whereas those which appear in the soluble fraction are stable. From these findings the conclusion is reached that certain enzymes of the endoplasmic reticulum are tightly bound to the membranes, whereas others either are loosely bound or are present in a soluble form within the lumina of the system. Some implications of these results as to the enzymic organization of the endoplasmic reticulum are discussed.