Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands.

Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.     

Ganglioside composition of subcellular fractions,including pre- and postsynaptic membranes,fromTorpedo electric organ

Gangliosides were isolated from four subcellular fractions of the electric organ ofTorpedo marmorata: synaptosomes, presynaptic membranes, postsynaptic membranes, and synaptic vesicle membranes. This exploited a principal advantage offered by this tissue: facile separation of pre-and postyynaptic elements. Total ganglioside concentration in presynaptic membranes was approximately twice that of synaptosomes and 15 times that of postsynaptic membranes (47.7, 24.4, and 3.21 g of lipid sialic acid per mg protein, respectively). Synaptic vesicle membranes had the highest overall concentration (78.9) relative to protein, but a concentration approximately comparable to that of presynaptic membranes when expressed relative to phospholipid. The thin-layer patterns of these two fractions were similar, both in terms of total pattern and the specific pattern of gangliotetraose structures as revealed by overlay with cholera toxin B subunit; these were notable for the paucity of monosialo structures and the virtual absence of GM1. Postsynaptic membranes, on the other hand, had a significantly higher content of monosialogangliosides including the presence of GM1. The synaptosomal pattern resembled that of the presynaptic membranes and synaptic vesicles. Thus, a clear difference in ganglioside pattern could be discerned between the pre- and postsynaptic elements of the electric organ.Abbreviations SVs synaptic vesicles - TLC thin-layer chromatography - cholera B-HRP B subunit of cholera toxin linked to horseradish peroxidase     

Effect of Denervation on a Cholinergic-Specific Ganglioside Antigen (Chol-1) Present in Torpedo Electromotor Presynaptic Plasma Membranes

The presence of Chol-1, an antigen identified in the plasma membrane of cholinergic electromotor nerve terminals of Torpedo marmorata, was investigated in Torpedo electric organ after 3, 6, and 9 weeks' denervation. Denervation was monitored by the cessation of stimulus-evoked discharge potentials, by the reduction in nerve terminals seen morphologically, and by the decrease in ACh and ChAT contents. The content of ganglioside-bound sialic acid did not show any appreciable change with time. Some modification of ganglioside pattern on TLC was observed after 9 weeks' denervation. The presence of Chol-1 after denervation was assayed by its activity in inhibiting the selective complement-induced lysis of the cholinergic subpopulation of guinea pig cortical synaptosome which is mediated by the anti-Chol-1 antiserum. Denervation did not affect Chol-1 immunoreactivity although it did alter the distribution of the immunoreactivity among gangliosides. The possible significance of the results is discussed.     

Gangliosides and Other Lipids of the Growth Cone Membrane

Growth cone membranes, derived from growth cone particles isolated from 16- to 18-day-old fetal rat brain, were found to be rich in overall lipid content with a lipid-to-protein ratio of 3.5. The phospholipid-to-cholesterol ratio indicated considerably less cholesterol than plasma membranes from mature neurons. All major classes of phospholipid were present in the usual proportions except sphingomyelin, which could not be detected. Gangliosides expressed in relation to protein were present at somewhat higher levels compared to previously reported values for synaptic plasma membranes (73 versus 44 micrograms/mg protein), but when related to phospholipid their level was well below that of the latter (26 versus 62 micrograms/mg phospholipid). The ganglioside pattern was generally similar to that of mature synaptic membranes except for the presence of relatively more GD3 and less GD1a, a phenomenon also observed in whole fetal brain of the same age. Several neutral glycosphingolipids were detected, glucosylceramide being the major one of this group. Their total level in growth cone membranes was roughly comparable to that of gangliosides, but unlike the latter their concentration in whole brain decreased with development. For comparison we analyzed the ganglioside composition of mixed membrane fractions from the same fetal brains and found no significant differences between these and growth cone membranes, suggesting that these glycoconjugates are not localized specifically in the growth cones. Neutral glycosphingolipids, on the other hand, appeared somewhat more concentrated in growth cones than in the mixed membranes.     

Transmembrane topology of acetylcholine receptor subunits probed with photoreactive phospholipids

The domains of the acetylcholine receptor subunits that contact the lipid phase were investigated by hydrophobic photolabeling of receptor-rich membrane fragments prepared from Torpedo marmorata and Torpedo californica electric organs. The radioactive arylazido phospholipids used carry a photoreactive group, either at the level of the lipid polar head group (PCI) or at the tip of the aliphatic chain (PCII), and thus probe respectively the "superficial" and "deep" regions of the lipid bilayer. The four subunits of T. marmorata and T. californica acetylcholine receptor reacted with both the PCI and PCII probes and thus are all exposed to the lipid phase. Ligands known to stabilize different conformations of the acetylcholine receptor (nicotinic agonists, snake alpha-toxin, and noncompetitive blockers) did not cause any significant change in the labeling pattern. The acetylcholine receptor associated 43 000-dalton v1 protein did not react with any of the probes. A striking difference in labeling between T. marmorata and T. californica acetylcholine receptors occurred at the level of the alpha-subunit when the superficial PCI probe was used. An approximately 5-fold higher labeling of the alpha-subunit as compared to the beta-, gamma-, and delta-subunits was observed by using receptor-rich membranes from T. marmorata but not from T. californica. The same difference persisted after purification of the labeled receptors from the two species and was restricted to an 8000-dalton C-terminal tryptic peptide. The only mutation observed in this region of the complete alpha-subunit sequence of the two species is the substitution of cysteine-424 in T. marmorata by serine-424 in T. californica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systematic position of fish species and ganglioside composition and content

The ganglioside content in brain of cartilaginous and bony fishes studied varies from 110 to 581 and from 104 to 595 micrograms sialic acid per g of wet weight respectively. A high degree of alkali lability and the predominance of C18-sphingosine and N-acetylneuraminic acid are typical of fish brain gangliosides. A high content of oligosialogangliosides with four and more residues of sialic acid and the predominance of gangliosides with gangliotetraosyl carbohydrate chain are characteristic for teleost brain. No pronounced difference was revealed in ganglioside composition and content of clupeomorphs and percomorphs. Gangliosides with short (lactosyl and gangliotriaosyl) carbohydrate chain predominate in brain of all cartilaginous fishes studied. A statistically significant difference was found in ganglioside content, relative oligosialoganglioside content and ganglioside fatty acid composition of squalomorphs and rajiformes, on one hand, and dasyatiformes and galeomorphs, on the other hand.     

Molecular weight of the acetylcholine receptors of electric organs and the effect of Triton X-100.

Studies are reported on the influence of Triton X-100 on the molecular weight and functional properties of the acetylcholine receptor. Results are presented principally for receptors purified from Torpedo californica and Torpedo marmorata with a limited number of observations on the receptor from Electrophorus electricus. In equilibrium dialysis measurements Trito, X-100 greatly reduced acetylcholine binding to the high affinity sites of the receptor from T. californica, but had only a small effect on the sites of lower affinity. Sedimentation equilibrium experiments on receptor in the absence of added Triton X-100 revealed average apparent molecular weight values of 510,000 for receptor from T. californica and 665,000 for T. marmorata. Under those conditions 0.113 mg of residual Triton X-100 were found per mg of protein as determined by using 3H-labeled Triton X-100. The sedimentation data indicated the presence of more than one molecular species, involving a unit with an apparent molecular weight of 330,000 and higher aggregates. Upon addition of Triton X-100, the higher aggregates were reduced, and above 0.1 percent Triton X-100 the 330,000 unit was the principal component present for receptor from all three species examined. Various structural models are considered in the light of this value, the polypeptide size from Na dodecyl sulfate-gel electrophoresis, and the protomer size determined by the molecular weight of an acetylcholine binding site.     

Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP1 synaptic vesicles banding at 1.055 g/mL actively transported [3H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [3H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP1 vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle originally described in intact Torpedo marmorata electric organ [Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 715-730]. The properties of the receptor for vesamicol were studied by measuring binding of [3H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP1 vesicles the VP2 vesicles had a ratio of [3H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. Based on an antibody standardization, in a typical preparation the VP1 vesicles contained 237 +/- 15 pmol of receptor/mg of protein whereas VP2 vesicles contained 102 +/- 3 pmol of receptor/mg of protein, and VP2 vesicles transported AcCh 2-3-fold more actively than VP1 vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ganglioside abnormalities associated with failed neural differentiation in a T-locus mutant mouse embryo

The T-locus on mouse chromosome 17 contains a number of mutations that disrupt cellular differentiation and embryonic development. Because of their purported role in neuronal differentiation and brain development, gangliosides were studied in mouse embryos homozygous for two T-locus mutations: T and twl. Mice homozygous for the dominant T mutation die from failed mesodermal differentiation in the notochord, whereas mice homozygous for the recessive twl mutation die from failed neural differentiation in the ventral portion of the neural tube. No major ganglioside abnormalities were found in T/T mutant embryos at Embryonic Day 10 (E-10). In contrast, E-11 twl/twl mutants expressed a marked deficiency of the tetrasialoganglioside GQ1. Since this ganglioside migrates with GQ1b in three different thin-layer solvent systems, it may have the same structure as GQ1b. To gain insight into regional distribution, gangliosides were examined in head regions and body regions of normal (+/+) E-11 embryos. The ganglioside composition of these regions was the same as that of the whole embryo, with GM3 and GD3 comprising about 75% of the total ganglioside distribution. Moreover, N-acetylneuraminic acid was the only sialic acid species detectable in the E-10 and the E-11 embryos. These findings indicate that N-acetylneuraminic acid-containing gangliosides are synthesized actively in E-10 and E-11 mouse embryos and also suggest that the GQ1 deficiency in the twl/twl mutants is closely associated with failed neural differentiation.     

Complex Carbohydrate Composition of Large Dense-Cored Vesicles from Sympathetic Nerve

Highly purified noradrenergic, large, dense-cored vesicles were isolated from bovine sympathetic nerve endings by sucrose-D2O density gradient centrifugation. Their concentration of glycoprotein hexosamine and sialic acid was 6.6 and 3.9 mumol/100 mg lipid-free dry weight, respectively, values which are similar to those previously found in bovine chromaffin granules. However, whereas chromaffin granule glycoproteins are characterized by their high proportion of N-acetylgalactosamine-containing O-glycosidically-linked oligosaccharides (present in the chromogranins), such oligosaccharides accounted for only 17% of those in noradrenergic synaptic vesicle glycoproteins. Fractionation of N-3H-acetylated glycopeptides by sequential lectin affinity chromatography demonstrated that approximately two-thirds of the oligosaccharides were of the tri- and tetraantennary complex type, accompanied by 14% biantennary oligosaccharides and 3% high-mannose oligosaccharides. The vesicles had a relatively low concentration of chondroitin sulfate (less than 5% of that in chromaffin granules) but significant amounts of heparan sulfate (0.4 mumol N-acetylglucosamine/100 mg lipid-free dry weight). No hyaluronic acid was detected. The concentration of ganglioside sialic acid in the noradrenergic vesicles was approximately 1 mumol/100 mg lipid-free dry weight, which is significantly higher than that of a crude membrane mixture from which the vesicles were prepared; the ratio of N-acetyl- to N-glycolylneuraminic acid was 0.8. Several molecular species of gangliosides were detected by thin-layer chromatography, but most of these did not exactly comigrate with bovine brain gangliosides. Cholera toxin binding indicated that approximately half or less of the gangliosides belong to the gangliotetraose series.     

Gangliosides and sialosylglycoproteins in coated vesicles from bovine brain.

The content of gangliosides and sialosylglycoproteins was investigated in a coated-vesicle-enriched fraction prepared from bovine brain by the method of Pearse [(1975) J. Mol. Biol. 97, 93-98] and further purified by g.p.c. (glass-permeation chromatography) [Pfeffer & Kelly (1981) J. Cell Biol. 91, 385-391]. From morphological criteria and from the analysis of the polypeptide pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the coated-vesicle fraction (CV-fraction) appeared more than 95% pure. The ganglioside-NeuAc (N-acetylneuraminate), glycoprotein-NeuAc, phospholipid and cholesterol contents of CV-fraction were compared with those of bovine brain synaptic plasma membranes (SPM). The cholesterol to phospholipid molar ratio was 0.47 +/- 0.07 in CV-fraction and 1.06 +/- 0.08 in SPM. The ganglioside-NeuAc and glycoprotein-NeuAc to phospholipid molar ratios were 0.047 and 0.020 respectively in CV-fraction and 0.039 and 0.016 respectively in SPM. The (Na+ + K+)-dependent ATPase activity sensitive to ouabain (in mumol of Pi/h per nmol of phospholipid) was 1.04 in CV-fraction and 0.63 in SPM; the ratio between this activity and the activity resistant to ouabain was 2 in CV-fraction and 1.4 in SPM. A t.l.c. analysis of the ganglioside fractions showed that most of the ganglioside species present in SPM were present in CV-fraction. In a rat brain coated-vesicle preparation not subjected to g.p.c., the activities [as sugar-radioactivity (c.p.m.) transferred/h per mumol of phospholipid] of the enzymes CMP-NeuAc:sialosyl-lactosylceramide (GM3) sialosyl-, UDP-Gal:N-acetylgalactosaminyl(sialosyl)lactosylceramide (GM2) galactosyl- and UDP-GalNAc:sialosyl-lactosylceramide (GM3) N-acetylgalactosaminyl-transferases, which were considered Golgi-apparatus markers, were about 19, 16 and 10% respectively of those determined in rat brain neuronal perikaryon-enriched fractions. Taken together, the results indicate that most of the major gangliosides are constituents of coated vesicles.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Putative Cholinergic-Specific Gangliosides in Guinea Pig Forebrain

The nature of the cholinergic-specific antigen Chol-1 recognized by an antiserum raised against Torpedo cholinergic electromotor synaptosomal plasma membranes was investigated in guinea pig forebrain to establish whether it has a gangliosidic nature in guinea pig as in Torpedo. Gangliosides extracted from guinea pig forebrain and extensively purified to eliminate peptide contaminants were effective in inhibiting the selective lysis of the cholinergic subpopulation of cortical synaptosomes induced by the antiserum. Neuraminidase, protease, alkali, and heat treatment did not impair the inhibitory activity of gangliosides. Whereas the antiserum recognized many gangliosides from Torpedo electric organ, the immunostaining of guinea pig forebrain gangliosides separated on TLC showed only two immunopositive bands migrating close to GT1b and GQ. After affinity purification on Torpedo electric organ gangliosides the activity of the antiserum in inducing complement-mediated lysis was increased and it still recognized the two ganglioside bands on TLC. These results strongly suggest the existence of two polysialogangliosides bearing antigenic determinants specific for the cholinergic neurons.     

Interactions between alpha-conotoxin MI and the Torpedo marmorata receptor alpha-delta interface

The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.     

GANGLIOSIDE COMPOSITION AND CONTENT OF RAT-BRAIN SUBCELLULAR FRACTIONS

Abstract— The composition and content of gangliosides from rat-brain microsomal, synaptosomal, mitochondrial and myelin fractions were studied. Outer membranes of synaptosomes were also isolated, separated into subfractions and investigated. Of all the fractions studied the outer membranes of synaptosomes are richest in gangliosides, in one of their sub-fractions the concentration of gangliosides per mg of protein is five times higher than in the homogenate. Microsomes are rich in gangliosides as well, but to a lesser degree, whereas the mitochondrial fraction contains considerably smaller amounts of gangliosides per mg of protein than does the homogenate. The ganglioside pattern of outer membranes of synaptosomes and of their subfractions is somewhat different from that of the homogenate; the outer membranes contain approximately one-third less monosialogangliosides. On the contrary a very high content of monosialogangliosides is characteristic of the ganglioside pattern of the myelin fraction. In this fraction monosialoganglioside G_MI (nomenclature of Svennerholm, 1963) constitutes 60–63 per cent of ganglioside sialic acid, or 75–80 molar per cent of gangliosides, the content of di- and trisialogangliosides being much lower than in other fractions. Fatty acid and long chain base composition of gangliosides from synaptosomal and microsomal fractions and homogenate is very similar, almost identical. In gangliosides from myelin fractions the relaitve content of palmitic and monoenoic acids is higher and that of arachinic acid and C₂₀-sphingosine—lower than in other fractions studied. The difference in ganglioside composition of synaptosomes and their outer membranes and on the other hand of myelin appears to reflect the difference in ganglioside composition of neuronal and oligodendroglial plasma membranes.     

Glycolipids modulate glycosyl transfer to endogenous protein acceptors

Regulation by gangliosides of glycosylation of endogenous membrane glycoproteins is indicated from in vitro studies in which incorporation of radioactive sugars into endogenous protein acceptors was measured and from in vitro studies where transferase activities of membranes were correlated with ganglioside content during hepatic tumorigenesis. Galactosyl transfer from UDP galactose exhibited a complex response pattern and was stimulated by lactosyl ceramide and the ganglioside N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM2) but was inhibited by higher gangliosides. Except for N-acetylneuraminylgalactosylglucosylceramide (GM3), which had no effect, inhibition was proportional to ganglioside complexity. Inhibition of glycosylation of the exogenous acceptor, ovomucoid, by ganglioside was slight by comparison. While marked structure-linked latency was observed with the high molecular weight exogenous acceptor, no latency was observed for incorporation into endogenous acceptors suggesting that the membranes were permeable to sugar nucleotides. Membrane disruption with detergents lessened rather than enhanced inhibition by gangliosides. Sialyl transfer from CMPsialic acid, on the other hand, was unaffected or stimulated by gangliosides. Stimulation by galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM1) was proportional to concentration and reached 2-fold at 240 micrograms/mg protein. The results suggest that the ganglioside content of membrane may affect glycosylation of membrane glycoproteins.     

Identification of a Synaptic Vesicle Antigen (Mr 86,000) Conserved Between Torpedo and Rat

Antisera were raised in guinea pigs to synaptic vesicles purified from the electric organ of Torpedo marmorata. In cholinergic nerve terminals from Torpedo the major antigens identified had Mr 300,000-150,000, 86,000, and 18,000. The Mr 86,000 antigen was conserved between Torpedo and rat, where it is neuron-specific and concentrated in nerve terminals. When rat brain synaptosomes are subfractionated the antigen is associated with synaptic vesicles. The antigen is not found in the cytoskeleton and in the vesicle-free cytosol. Immunohistochemical localization of the antigen in rat shows it to be associated with synapses in diaphragm, cerebellum, hippocampus, and cerebral cortex. The staining pattern of the antigen indicates that the antigen is not cholinergic-specific. The function of the Mr 86,000 antigen remains to be identified.     

Composition of Lipids in Elasmobranch Electric Organ and Acetylcholine Receptor Membranes

The composition of phospholipids from electric organ and from membranes enriched in acetylcholine receptors (AChRs) is analyzed in three elasmobranch fish (Torpedo marmorata, Torpedo californica, and Discopyge tschudii). Irrespective of their purity, AChR-containing membranes are similar to electric organ in lipid and fatty acid composition. The following characteristics are common to the three species: (a) Choline, ethanolamine, and serine glycerophospholipids account for 80-90% of the phospholipids. (b) Their major fatty acid constituents are monoenes, saturates, and long-chain (n-3) polyenes (especially docosahexaenoate). (c) A large proportion of the ethanolamine glycerophospholipids (30-50%) is made up by plasmenylethanolamine, which contains fewer polyenes than phosphatidylethanolamine per mole of lipid. (d) Polyphosphoinositides represent 20-30% of the inositides of electric organ. (e) Phosphatidylinositol and phosphatidate have large proportions of 20- and 22-carbon polyenes. (f) Diphosphatidylglycerol and triacylglycerols are rich in oleate but also contain long-chain polyenes. (g) Sphingomyelin has monoenes and saturates ranging from 14 to 26 carbons. Species-related variations are observed (a) in the ratios between some phospholipid classes and subclasses and (b) in the relative abundance of the major polyunsaturated acyl chains of phospholipids. Despite these differences, the average unsaturation and length of fatty acids in major phospholipid classes are similar for the three species.     

The presence of sialidase-sensitive sialosylgangliotetraosyl ceramide (GM1b) in stimulated murine macrophages. Deficiency of GM1b in Escherichia coli-activated macrophages from the C3H/HeJ mouse

The stimulated murine macrophage was found to contain 11 major gangliosides of which 8 were determined to be monosialylated. The thin-layer chromatographic patterns were complicated by the presence of both sialic acid and ceramide fatty acid heterogeneity. N-glycolyl and N-acetylneuraminic acid-containing species were present for each ganglioside characterized. Although C18 sphingosine was the only long chain base detected, ceramide fatty acid ranged from C16 to C24 carbon moieties. Based on gas-liquid chromatographic and antibody analyses, all major tetraosyl structure gangliosides were ganglio series types. Comprising 43 to 60% of thioglycollate-stimulated cells and 60 to 70% of Escherichia coli-activated cells, monosialosyl-gangliotetraosyl ceromides (Gm1 gangliosides) were the major monosialo species of which four were present: sialidase-resistant NeuGc-GM1a and NeuAc-GM1a and sialidase sensitive NeuGc-GM1b and NeuAc-GM1b. Analyses of thioglycollate-elicited murine peritoneal macrophage ganglioside patterns from four strains of mice, including the C3H/HeJ strain, indicated that, in the absence of any expression of a genetic defect, the pattern is conserved. However, when E. coli was used as the activating agent, the normal C3H/HeN macrophage contained little Gm1a with the sialidase-sensitive Gm1b predominant; the converse was true for the congenic endotoxin hyporesponsive C3H/HeJ strain. Therefore, C3H/HeJ mice are not defective in ganglioside metabolism per se but in the processing of an endotoxin stimulus such that one manifestation is an altered macrophage ganglioside pattern deficient in Gm1b.     

A Ganglioside Species (GD1α) Migrates at a Slow Rate and CMP-Sialic Acid Severalfold Faster in Xenopus Sciatic Nerve: Fluorographic Demonstration

The ninth dorsal root ganglion of adult Xenopus laevis was labeled with N-acetyl-D-[6-3H]mannosamine, and intraaxonal migration of gangliosides was examined by analysis of the chloroform/methanol extract of each of 5-mm consecutive nerve segments by TLC coupled with fluorography. A unique disialoganglioside (GD1 alpha), which amounted to up to 83% of the total ganglioside in this nerve, migrated at 1-2 mm/day at 15 degrees C. This contrasts with the rapid transport of other ganglioside species previously reported in the optic systems of goldfish, rabbits, chickens, and rats. Fluorographic analysis also revealed a trichloroacetic acid-soluble substance migrating at a velocity of approximately 8 mm/day at 15 degrees C. The substance was considered to be CMP-sialic acid on the basis of observations that it comigrates with authentic CMP-N-acetylneuraminic acid in TLC developed with two different solvent systems, it is very labile to weak acid but resistant to neuraminidase from Vibrio cholerae, it is converted to N-acetylmannosamine when treated first with weak acid and subsequently with N-acetylneuraminic acid aldolase, and it has a beta-sialosyl group in its structure. Because CMP-sialic acid is believed to be the sole sialosyl donor in the cells, its migration in axons toward terminals, together with the previous demonstration of sialyltransferase activity in the synaptosomal plasma membrane, strongly supports the possibility that sialosylation of gangliosides and probably of other sialoglycoproteins is not confined to the Golgi apparatus, but can also occur after the compounds are committed to axonal transport.