Effects of Brefeldin A on Synthesis and Intracellular Transport of Ganglioside GT3 by Chick Embryo Retina Cells

Abstract: Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [³H]galactose in the absence (control cells) or in the presence of 1 µg/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C . perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.     

Isolation of a domain of the plasma membrane in Chinese hamster ovary cells which contains iodinatable, acidic glycoproteins of high molecular weight

A light vesicle fraction, apparently derived from the plasma membrane, was obtained following breakage of Chinese hamster ovary (CHO) cells by means of a fluid pump disrupting device. The final preparation was enriched approx. 40-fold over the homogenate in K+,Na+-stimulated ATPase and phosphodiesterase I, but only approx. 10-fold in 125I specific radioactivity after lactoperoxidase-catalyzed iodination. This preparation was compared with another plasma membrane fraction purified as large sheets via a two-phase centrifugation procedure. Two-dimensional polyacrylamide gel electrophoresis followed by Coomassie blue staining indicated that both fractions were fairly similar in polypeptide composition, although a few consistent differences were evident. However, staining of glycoproteins by the periodic acid-Schiff technique or by overlaying with 125I-labeled concanavalin A showed that the vesicle fraction was highly enriched in groups of high molecular weight, acidic glycoproteins which stain only weakly with Coomassie blue. These glycoproteins also bound 125I-labeled ricin I agglutinin and wheat germ agglutinin. They appear to be the major receptors for wheat germ agglutinin on the CHO cell surface. After surface labeling of cells by the 125I-lactoperoxidase technique, the membrane sheet fraction contained a large number of iodinated polypeptides, whereas labeling in the vesicle fraction was restricted almost entirely to the high molecular weight, acidic glycoproteins. It is proposed that the vesicle fraction constitutes a specific domain of the cell surface which is coated on its exterior by this group of glycoproteins. These components probably mask underlying proteins of the plasma membrane from external labeling.     

Inhibition by Forskolin of Excitatory Amino Acid-Induced Accumulation of Cyclic AMP in Guinea Pig Hippocampal Slices

Left sciatic nerves of adult male Sprague-Dawley rats were crushed and allowed to recover for 0, 1, 2, 4, 7, or 14 days. At each of these times both L-5 dorsal root ganglia were injected with 100 microCi of [3H]glucosamine. Two days later, dorsal root ganglia, lumbosacral trunks, and sciatic nerves were removed bilaterally. The amounts of radiolabelled ganglioside in crushed lumbosacral trunks were consistently higher than in the controls, with the largest difference occurring within 2 days from simultaneous crush and injection to killing (specimens labelled day 0). The largest difference in the amount of radiolabelled ganglioside between crushed and control sciatic nerve (4-9 days from crush to killing) occurred later than that of lumbosacral trunk, but no significant difference occurred within the first 3 days following crush. There was only a slightly higher radioactivity in gangliosides totalled from all three anatomical specimens of crushed than in control nerves. The neutral nonganglioside lipid and acid-precipitable fraction followed patterns of synthesis and accumulation similar to those of the gangliosides. These findings indicate that after nerve crush gangliosides, glucosamine-labelled neutral nonganglioside lipids, and glycoproteins accumulate close to the proximal end of the regenerating axon. This accumulation could serve as a reservoir to increase the ganglioside concentration in the growth cone membrane.     

Entry of Newly Synthesized Gangliosides into Myelin

Ganglioside synthesis and transport to myelin was studied in brainstem slices prepared from 19-21-day-old rats. The slices were incubated for up to 2 h in the presence of [3H]glucosamine to label primarily the hexosamine portion of complex gangliosides. The amount of radioactivity incorporated into gangliosides during slice incubations was only 10-15% of the amount of the label incorporated during in vivo labeling of brainstem gangliosides using equivalent amounts of [3H]glucosamine. Among individual gangliosides this inhibition was greater for the more complex gangliosides. When labeled gangliosides were isolated from homogenate and myelin fractions prepared from brain slices, the complex total gangliosides of both fractions showed a lag in labeling kinetics but with a lower specific radioactivity for the myelin fraction, reflecting the larger pool size and slower turnover rate exhibited by myelin components. Chase experiments showed that more complex gangliosides in homogenate exhibited almost no effect of chase after 30 min. Addition of the Golgi-disrupting agent monensin to slice incubations inhibited the labeling of all gangliosides except GM3, GM2, and GD3, and transport to myelin of all complex gangliosides except GM2. These results show that a monensin-sensitive mode of transport is responsible for the translocation of most newly synthesized gangliosides into myelin.     

Disposition of Gangliosides and Sialosylglycoproteins in Neuronal Membranes

Abstract Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP-[³H] N -acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [³H] sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 m -KCl, 125 m m -EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of the neuraminidase-labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase-labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labeled in vivo by an intracerebral injection of N -[³H]acetylmannos-amine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with the behavior of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.     

A study of intracellular transport of secretory glycoproteins in rat liver

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.     

Incorporation of N-Acetylmannosamine into Rat Brain Subcellular Gangliosides: Effect of Pentylenetetrazol-Induced Convulsions on Brain Gangliosides1

Abstract: The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N-acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides G_T1b and G_Q1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, G_T1b and G_Q1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane-bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water-soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin-layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin-layer chromatogram.     

A part of glucosylceramide formed from exogenous lactosylceramide is not degraded to ceramide but re-cycled and glycosylated in the Golgi apparatus

The subcellular fate of glucosylceramide (GlcCer) formed from exogenous lactosylceramide (LacCer) in rat liver is investigated. LacCer radiolabeled on different positions of the molecule was intravenously administered to rats as a liposomal dispersion. A Golgi apparatus fraction 140-fold enriched in specific markers and constituted by intact cisternal stacks, as well as the lysosomal and plasma membrane fractions concurrently prepared from the same homogenate, were then studied in order to determine the time course of radioactive glycosphingolipids. LacCer quickly decreased with time in the plasma membrane, whereas in the lysosomes it increased up to 4 h and decreased thereafter. In both fractions results were regardless of the labeling position. In the Golgi apparatus, LacCer increased up to 12 h and then decreased. In this fraction, the radioactivity values of [Glc-3H]LacCer were over twice those of [Gal-3H]LacCer. GlcCer was found only after [Glc-3H]LacCer administration. In the lysosomes, its time course provided a peak similar in shape but delayed in timing with respect to that of LacCer. Conversely, in the Golgi apparatus GlcCer was earlier formed, but earlier consumed, than LacCer. Gangliosides increased in the Golgi apparatus until 4 h and then decreased after 12 h, whereas in the plasma membrane they were progressively accumulated. In both fractions the amount of [Glc-3H]gangliosides was over twice that of [Gal-3H]gangliosides was over twice that of [Gal-3H]gangliosides. Since we demonstrated that the sugars released in the course of LacCer degradation (LacCer----galactose + GlcCer----glucose + ceramide) are not incorporated into glycoconjugates, we conclude that a part of GlcCer formed during the lysosomal degradation of LacCer actually reaches the Golgi apparatus where it undergoes successive glycosylation.     

Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver.

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.     

Electron paramagnetic resonance studies on the fluidity and surface dynamics of egg phosphatidylcholine vesicles containing gangliosides

The influence of different gangliosides (G_M1, G_D1a, G_T1b) on the fluidity and surface dynamics of phosphatidylcholine small unilamellar vesicles was studied by electron paramagnetic resonance. 5-and 16-nitroxystearic acid, sounding respectively the region close to the surface and that close to the hydrophobic core of the vesicle, were employed as spin-label probes. The signals released by 5-nitroxystearic acid showed that the presence of gangliosides reduced the mobility of the hydrocarbon chains around the probe. The effect increased by increasing ganglioside concentration, and diminished from G_M1 to G_D1a and G_T1b. The decrease of membrane fluidity was also monitored by the 16-nitroxystearic acid probe. On addition of Ca²⁺ the fluidity of ganglioside-containing vesicles (as signalled by the 5-nitroxystearic acid probe) promptly decreased, thereafter returning slowly to the original value. It is suggested that gangliosides cause strong side-side head group interactions on the bilayer surface -between ganglioside oligosaccharide chains and between ganglioside and phosphatidylcholine polar portions - which lead the lipid chains to assembly in a more rigid fashion. The influence of Ca²⁺ is interpreted as due to lateral phase separation in the vesicle membrane. This phenomenon can be related to the formation or stabilization of ganglioside clusters on the vesicle surface.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

Incorporation of [14C]glucosamine into synaptosomes in vitro

Abstract— Synaptosomes isolated from rat cerebral cortex by zonal centrifugation in-corporated radioactive glucosamine into macromolecules in vitro as glucosamine, galactosamine, N-acetylneuraminic acid, and glucuronic acid. The largest percentage of incorporated radioactivity was recovered in the particulate fraction in which radioactive carbohydrates were bound in covalent linkage requiring acid hydrolysis or enzymatic digestion for release. Less than 20 per cent of the particulate radioactivity represented incorporation into gangliosides. Some 20 per cent of the radioactivity was incorporated into proteins as glucosamine, identified in hydrolysates by paper chromatography and by the amino acid analyser. After incubation, radioactivity was demonstrable in the proteins as sialic acid by paper chromatography and specific enzymic digestion; and as glucuronic acid by chromatography, electrophoresis, and digestion with hyaluronidase. Incorporation of carbohydrate was stimulated by sodium and potassium at concentrations demonstrated to enhance incorporation of amino acids, and involved the macro-molecules of all subsynaptosomal fractions. Significant incorporation of radioactivity was found in the synaptic plasma membrane. The synthesis of glycoproteins was suggested by simultaneous incorporation of [¹⁴C]glucosamine and [³H]leucine into glycopeptides subsequently hydrolysed and subjected to polyacrylamide gel electrophoresis and two-dimensional paper chromatography and electrophoresis. Such studies demonstrated that amino acids and carbohydrates may be incorporated into glycoproteins of the synaptic membrane and suggest the possibility of local synthesis as well as modification of material brought to the nerve ending by axoplasmic flow.     

Inhibition of Kinesin Synthesis In Vivo Inhibits the Rapid Transport of Representative Proteins for Three Transport Vesicle Classes into the Axon

Abstract: We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide-containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.     

Distribution of [3H]Dihydrotetrabenazine Binding in Bovine Striatal Subsynaptic Fractions: Enrichment of Higher Affinity Binding in a Synaptic Vesicle Fraction

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3-9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95-400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.     

Uptake, cell penetration and metabolic processing of exogenously administered GM1 ganglioside in rat brain

GM1 ganglioside, after intravenous injection into rats, is absorbed and taken up by various organs and tissues, including brain. The capacity of brain to take up gangliosides, referred to weight unit, is comparable to that of kidney and muscle. After injection of [Gal-³H]GM1 a relevant portion of brain associated radioactivity resided in the soluble fraction and was of a volatile nature. After brain subcellular fractionation, the lysosomal, plasma membrane and Golgi apparatus fractions carried the highest specific radioactivity. In addition, an enriched fraction of brain capillaries was highly labelled, suggesting that GM1 ganglioside is also tightly bound to the vessel walls.

The metabolic events encountered in brain by exogenous gangliosides were investigated, in detail, after intracisternal injection of [Sph-³H]GM1. The results obtained demonstrate that GM1 is extensively metabolized in brain. Besides the degradation products (GM2, GM3, lactosylceramide, glucosylceramide, ceramide), compounds of a biosynthetic origin were also found to be formed: these include GD1a, GD1b and sphingomyelin.

All the above results could indicate that gangliosides, after intravenous administration to rats, are taken up by brain, bind to the capillary network, penetrate into neural cells, associate to both plasma membranes and intracellular structures and undergo metabolic processing with formation of a number of products of both catabolic and biosynthetic origin.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.     

Characterization of a secretory vesicle-rich fraction from lactating bovine mammary gland.

Fractions enriched in secretory vesicles were obtained from lactating bovine mammary tissue by a straightforward procedure involving gentle homogenization and centrifugation in isotonic milk salt solution containing Ficoll. Secretory vesicle-rich fractions could also be obtained from lactating rat mammary gland by this procedure. With rats, yields of vesicles were substantially increased by administration of colchicine or thioglucose to animals several hours before sacrifice. Isolated fractions were enriched in lactose and consisted predominantly of 0.2–1.2 μm diameter vesicles, many of which contained casein micelles. Enzymatic, compositional and morphological examination revealed vesicle preparations to be largely free of contamination by rough endoplasmic reticulum, mitochondria, nuclei, peroxisomes and lysosomes. Specific activity of several marker enzymes of the secretory vesicle fraction were similar to, or intermediate between, Golgi apparatus and milk lipid globule membranes. Amounts of cholesterol and gangliosides in vesicle fractions approached levels found in plasma membranes. In distribution of major phospholipids, secretory vesicles were intermediate between Golgi apparatus and milk lipid globule membranes. The pattern of polypeptides of secretory vesicle membrane was qualitatively similar to that of Golgi apparatus membranes. While there were similarities between these polypeptide patterns and that of lipid globule membranes, the latter contained relatively more of certain polypeptides, particularly the internal coat-associated polypeptides of the globule membrane. These observations are discussed in relation to the endomembrane hypothesis and the origin of the membrane of milk lipid globules.     

Recycled Synaptic Vesicles Contain Vesicle but Not Plasma Membrane Marker, Newly Synthesized Acetylcholine, and a Sample of Extracellular Medium

To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low-frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate-dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extracellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low-frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.     

Short term labeling of proteins,gangliosides and glycoproteins of the optic tract of chickens exposed to light or darkness

In contrast with previous findings of the labeling of the glycosidic moieties of the gangliosides and glycoproteins in chickens injected with $\text{N-[}{}^3\text{H}\text{]}\text{acetylmannosamine}$, the labeling of the ganglion cell layer and optic tectum proteins of chicks exposed to light after an intraocular injection of [³H]proline showed no differences with those of their counterpart chickens that remained in darkness. The same failure in finding a difference was met when the cytosolic or the particulate proteins or the acid soluble fraction in the retina were compared.Cycloheximide and puromycin inhibited the labeling of retina and optic tectum proteins, gangliosides and glycoproteins in both illumination conditions. Since the labelings in the optic tectum appeared more inhibited than those in the retina ganglion cell layer it was concluded that cycloheximide and puromycin, besides the synthesis of those compounds, also inhibit their axonal transport.On the basis of these contrasting results the working hypothesis is advanced that light stimulation enhances the activity of the Golgi apparatus but not (or less) that of the polyribosomes.