Form of the postsynaptic density. A serial section study.

Through the use of serial sectioning of dog cerebral cortex tissue, holes or perforations could be revealed in the larger postsynaptic densities (PSDs), in confirmation of the earlier work of Peters and Kaisermann-Abramof (1969. Z. Zellforsch. Mikrosk. Anat. 100:487-506). These holes appeared in serial sections which happened to be cut both parallel and normal to the plane of the synaptic junction. Cleft material was absent in that part of the synaptic cleft opposite this hole. Somestimes the presynaptic membrane opposite the hole was indented into the presynaptic cell. In addition, most of the synaptic vesicles in the presynaptic cell close to the membrane were clustered at that part of the membrane opposite the edge of the density disk. The meaning of the hole and of the other features mentioned above for the function of the density is not known at present.

Distribution of newly synthesized amylase in microsomal subfractions of guinea pigs pancreas.

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).(1) Amylase appeared in the following positions in the gradient: (a) a light region which contained approximately 35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained approximately 10% of enzymic activity in a sharp peak but which had very little accompanying OD(260) absorption; (c) a pellet at the bottom of the centrifuge tube which contained approximately 20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C(14), radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized approximately 80% of the total amylase in the fraction from the heavy region of the gradient, but only approximately 20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations.

A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X-100, has been found to be identical to fodrin (Levine, J., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-125I-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido-calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin.

Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a dark-grown algal mutant (Chlamydomonas reinhardi).

This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.

Biogenesis of chloroplast membranes. II. Plastid differentiation during greening of a dark-grown algal mutant (Chlamydomonas reinhardi).

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6-8 hr in the absence of cell division. Photosynthetic activity (O(2) evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.

Biogenesis of endoplasmic reticulum membranes. I. Structural and chemical differentiation in developing rat hepatocyte.

The development of the endoplasmic reticulum of rat hepatocytes was studied during a period of rapid cell differentiation, i.e., from 3 days before to 8 days after birth. Before birth, the ER increases in volume, remaining predominantly rough surfaced; after birth, the increase continues but affects mainly the smooth-surfaced part of the system. These changes are reflected in variations of the RNA/protein and PLP/protein ratios of microsomal fractions: the first decreases, while the second increases, with age. The analysis of microsomal membranes and of microsomal lipids indicates that the PLP/protein ratio, the distribution of phospholipids, and the rate of P(32) incorporation into these phospholipids show little variation over the period examined and are comparable to values found in adult liver. Fatty acid composition of total phosphatides undergoes, however, drastic changes after birth. During the period of rapid ER development in vivo incorporation of leucine-C(14) and glycerol-C(14) into the proteins and lipids of microsomal membranes is higher in the rough-than in the smooth-surfaced microsomes, for the first hours after the injection of the label; later on ( approximately 10 hr) the situation is reversed. These results strongly suggest that new membrane is synthesized in the rough ER and subsequently transferred to the smooth ER.

Biogenesis of endoplasmic reticulum membranes. II. Synthesis of constitutive microsomal enzymes in developing rat hepatocyte.

The constitutive enzymes of microsomal membranes were investigated during a period of rapid ER development (from 3 days before to 8 days after birth) in rat hepatocytes. The activities studied (electron transport enzymes and phosphatases) appear at different times and increase at different rates. The increase in the enzyme activities tested was inhibited by Actinomycin D and puromycin. G-6-Pase and NADPH-cytochrome c reductase activities appeared first in the rough microsomes, and subsequently in smooth microsomes, eventually reaching a uniform concentration as in adult liver. The evidence suggests that the enzymes are synthesized in the rough part, then transferred to the smooth part, of the ER. Changes in the fat supplement of the maternal diet brought about changes in the fatty acid composition of microsomal phospholipids but did not influence the enzymic pattern of the suckling. Microsomes from 8-day-old and adult rats lose 95% of PLP and 80% of NADH-cytochrome c reductase activity after acetone-H(2)O (10:1) extraction. However, one-half the original activity could be regained by adding back phospholipid micelles prepared from purified phospholipid, or from lipid extracts of heart mitochondria, or of liver microsomes of 8-day or adult rats, thus demonstrating an activation of the enzyme by nonspecific phospholipid. The results suggest that during development the enzymic pattern is not influenced by the fatty acid or phospholipid composition of ER membranes.

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ.

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : II. Glucose-6-Phosphatase in Rough Microsomes.

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.

Synthesis and turnover of ribulose biphosphate carboxylase and of its subunits during the cell cycle of Chlamydomonas reinhardtii.

The chloroplast enzyme ribulose-1,5-bisphosphate (Ru-1,5-P2) carboxylase (EC 4.1 1.39) is made up ot two nonidentical subunits, one synthesized in the chloroplast and the other outside. Both of these subunits of the assembled enzyme are synthesized in a stepwise manner during the synchronous cell cycle of the green alga Chlamydomonas reinhardtii. The activity of this enzyme increases in the light and this increase is due to de novo protein synthesis as shown by the measurement of the amount of protein and by the pulse incorporation of radioactive arginine in the 18S enzyme peak in linear sucrose density gradients. During the dark phase of the cell cycle, there is little change in the enzymatic activity as well as in the amount of this enzyme. Pulse-labeling studies using radioactive arginine indicated that there is a slow but detectable rate of synthesis of the carboxylase and of its subunits in the dark. Ru-1,5-P2 carboxylase, prelabeled with radioactive arginine throughout the entire light period, shows a similarly slow rate of degradation in the following dark period. This slow turnover of the enzyme in the dark accounts for the steady levels of carboxylase protein and of enzymatic activity during this period. A wide variety of inhibitors of protein synthesis by 70S and 80S ribosomes abolished the incorporation of [3H]arginine into total Ru-1,5-P2 carboxylase during short-term incubation. These results suggest a tight-coordinated control of the biosynthesis of the small and large subunits of the enzyme. This stringent control is further substantiated by the finding that both subunits are synthesized in sychrony with each other, that the ratio of radioactivity of the small to the large subunit remains constant throughout the entire light-dark cycle, and that the rates of synthesis and of degradation of both subunits are similar to that of the assembled enzyme.

Function of calmodulin in postsynaptic densities. I. Presence of a calmodulin-activatable cyclic nucleotide phosphodiesterase activity.

The postsynaptic density (PSD) fraction from canine cerebra cortex was found to contain an endogenous cyclic nucleotide-phosphodiesterase activity that was independent on Mn2+ and/or Mg2+ but not on Ca2+. Maximal activity was obtained at 1 micrometer Mn2+. This cyclic nucleotide phosphodiesterase activity was not decreased upon removal of the calmodulin from the PSD fraction, nor was it increased by the addition of calmodulin to a postsynaptic density fraction deficient in calmodulin. The enzymatic activity could be extracted by sonication, with the soluble enzyme having properties similar to those found in the native structure. Two peaks of cyclic nucleotide phosphodiesterase activities could be obtained after S-300 Sephacryl column chromatography of this soluble fraction: fraction I (excluded peak) and fraction II (215,000 mol wt). The fraction I activity preferred cyclic AMP over cyclic GMP and was not activated by calmodulin. The fraction II activity has an approximately fourfold lower Km for cyclic GMP over cyclic AMP. This fraction II activity was activatable by calmodulin, which increased the Vmax and decreased the Km in the case of both cyclic nucleotides. We conclude that two activities are present in the PSD, one activatable, and one not activatable, by calmodulin.

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density.

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.

Function of a calmodulin in postsynaptic densities. II. Presence of a calmodulin-activatable protein kinase activity.

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [gamma-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [gamma-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.

Changes in chemical composition of thylakoid membranes during greening of the y-1 mutant of Chlamydomonas reinhardi.

Two fractions of thylakoid membranes (TMF) have been isolated from disrupted (French press) algal cells by using a discontinuous sucrose gradient. TMF-II consists mostly of thylakoid membranes still partially organized in grana; it contains also fragments of chloroplast envelope, pyrenoid tubules, and starch granules; thus it amounts to a fraction of chloroplast fragments which have lost practically all matrix components. TMF-I consists of smaller chloroplast fragments and is contaminated to a larger extent than TMF-II by other subcellular components, primarily mitochondria. TMF-II accounts for about 12% of the protein and 30% of the chlorophyll of the whole cell; it contains cytochrome 554 and carotenoids in the same ratio to chlorophyll as the latter, and shows photosystems I and II activities but lacks enzymatic activities characteristic of the dark reactions. During the greening of the y-1 mutant of Chlamydomonas, TMF's have been isolated over a range of chlorophyll concentrations from 5 to 25 microg/10(7) cells. The results showed that during this period the ratios of chlorophyll to cytochrome 554 and of chlorophyll to carotenoids, and the relative concentrations of individual carotenoids were continuously changing. The findings support the view that during greening, thylakoid membranes are produced by multistep assembly.

Isolation of cytoplasmic and chloroplast ribosomes and their dissociation into active subunits from Chlamydomonas reinhardtii.

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10-40% sucrose gradient containing 25 mM KCl and 5 mM MgCl(2). Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5-20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [(14)C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.

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

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is 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 in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ 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 approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. 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- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii.

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.

The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition.

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of 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-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids 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 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 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 or 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 (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.

Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.

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, J. 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 approximately 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 Mr bands were phosphorylated. In the PSDs from all brain regions, cAMP stimulated the phosphorylation of Protein Ia (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 the type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed.

Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization.

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes approximately 90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at approximately 13 x 10(6)g.min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation.