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.     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

Cerebellar granule cells in culture exhibit a ganglioside-sialidase presumably linked to the plasma membrane

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GD1a in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GM1, product of GD1a desialosylation. No GM1 or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine, or GD1a endocytosis prevented at 4 degrees C, the only metabolite formed was GM1. These results suggest that GD1a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H-Gal]GD1b, whereas no degradation of [3H-NeuAc]GM1 took place in the presence of chloroquine or at 4 degrees C. In conclusion, cerebellar granule cells express in vivo a sialidase, presumably located on the cell surface, that affects GD1a and GD1b but not GM1.     

Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture.

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.     

Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes.

We have made a comprehensive and quantitative analysis of the lipid composition of caveolae from primary rat fat cells and compared the composition of plasma membrane inside and outside caveolae. We isolated caveolae from purified plasma membranes using ultrasonication in carbonate buffer to disrupt the membrane, or extraction with nonionic detergent, followed by density gradient ultracentrifugation. The carbonate-isolated caveolae fraction was further immunopurified using caveolin antibodies. Carbonate-isolated caveolae were enriched in cholesterol and sphingomyelin, and the concentration was three- and twofold higher, respectively, in caveolae compared to the surrounding plasma membrane. The concentration of glycerophospholipids was similar suggesting that glycerophospholipids constitute a constant core throughout the plasma membrane. The composition of detergent-insoluble fractions of the plasma membrane was very variable between preparations, but strongly enriched in sphingomyelin and depleted of glycerophospholipids compared to carbonate-isolated caveolae; indicating that detergent extraction is not a suitable technique for caveolae preparation. An average adipocyte caveola contained about 22 x 10(3) molecules of cholesterol, 7.5 x 10(3) of sphingomyelin and 23 x 10(3) of glycerophospholipid. The glycosphingolipid GD3 was highly enriched in caveolae, whereas GM3, GM1 and GD1a were present inside as well as outside the caveolae membrane. GD1b, GT1b, GM2, GQ1b, sulfatide and lactosylceramide sulfate were not detected in caveolae.     

On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. III. Isolation of an intermediate in the biosynthesis of biotin from dethiobiotin.

A plasma membrane fraction more than 10-fold enriched in 5′-nucleotidase and alkaline phosphatase was prepared from peritoneal polymorphonuclear leukocytes. This fraction was highly enriched in MgATPase and a b-type cytochrome. High NADPH-duroquinone reductase activity was observed in the leukocytes, in addition to a lipid with quinone-like properties, neither of which cofractionated with plasma membrane. Therefore, we propose the possibility that an electron transport chain which functions to produce microbicidal oxygen metabolites is subdivided between the plasma membrane and one of the cytoplasmic membranes in non-phagocytizing cells.     

Effect of gangliosides on the distribution of a glycosylphosphatidylinositol-anchored protein in plasma membrane from Chinese hamster ovary-K1 cells

Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.     

Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells*

Gangliosides of the plasma membrane are important modulatorsof cellular functions. Previous work from our laboratory hadsuggested that a plasma membrane sialidase was involved in growthcontrol and differentiation in cultured human neuroblastomacells (SK-N-MC), but its substrates had remained obscure. Wenow performed sialidase specificity studies in subcellular fractionsand found ganglioside GM3 desialylating activity in presenceof Triton X-100 to be associated with the plasma membrane, butabsent in lysosomes. This Triton-activated plasma membrane enzymedesialylated also gangliosides GDla, GD1b, and GT1b, therebyforming GM1; cleavage of GM1 and GM2, however, was not observed.Sialidase activity towards the glycoprotein fetuin with modifiedC-7 sialic acids and towards 4-methylumbelliferyl neuraminatewas solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in ganglioside desialylationof living cells was examined by following the fate of [³H]galactose-labelledindividual gangliosides in pulse-chase experiments in absenceand presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminicacid. When the plasma membrane sialidase was inhibited, radioactivityof all gangliosides chased at the same rate. In the absenceof inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degradedat a considerably faster rate in confluent cultures, whereasthe GM1-pool seemed to be filled by the desialylation of highergangliosides. The results thus suggest that the plasma membranesialidase causes selective ganglioside desialylation, and thatsuch surface glycolipid modification triggers growth controland differentiation in human neuroblastoma cells. ganglioside neuroblastoma cells plasma membrane sialidase     

Rapid internalization of exogenous ganglioside GM3 and its metabolism to ceramide in human myelogenous leukemia HL-60 cells compared with control ganglioside GM1

Incorporation and metabolism of exogenous GM3 in human myelogenous leukemia HL-60 cells were analyzed using ³H-labeled GM3 ([³H]GM3). [³H]GM3 was rapidly internalized into the cells (trypsin-resistant fraction) 8 times more than the control, ³H-labeled GM1 ([³H]GM1). In addition, not only incorporation but also metabolism of [³H]GM3 was more rapid than [³H]GM1 in HL-60 cells. Moreover, one of the metabolites was found to co-migrate with ceramide in thin-layer chromatography analysis and ceramide formation from exogenous GM3 is more rapid than that from exogenous GM1. These results suggested that there would be some preferential mechanism to produce ceramide from differentiation-inducible GM3 in HL-60 cells rather than from non-inducing GM1.     

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.     

Lysosomal and plasma membrane ganglioside GM3 sialidases of cultured human fibroblasts. Differentiation by detergents and inhibitors

Cultured human fibroblasts contain two sialidases that degrade gangliosides such as GM3: a lysosomal activity that appears identical with the activity towards water-soluble substrates and that is deficient in the genetic lysosomal disorder sialidosis, and another enzyme that seems localized on the external surface of the plasma membrane. In this report we show that both enzymes can be differentiated in the presence of each other by choice of the detergent used for activation, and also by the inhibitory action of some polyanionic compounds such as sulphated glycosaminoglycans. The lysosomal ganglioside GM3 sialidase is greatly stimulated by sodium glycodeoxycholate and, to lesser degrees, by sodium glycocholate and sodium cholate. The ganglioside GM3 sialidase of the plasma membrane is not measurably active under the conditions of the lysosomal enzyme but is specifically activated by the non-ionic detergent Triton X-100. The glycodeoxycholate-stimulated, but not the Triton-activated, ganglioside GM3 sialidase activity was profoundly diminished in cell lines from patients with the lysosomal disorders sialidosis and galactosialidosis; however, both activities were normal in fibroblasts from patients with mucolipidosis IV, previously thought to be a ganglioside sialidase deficiency disorder. Both the lysosomal and the plasma membrane ganglioside GM3 sialidases were inhibited by sialic acids, suramin, dextran sulphate and sulphated glycosaminoglycans. Among the latter, heparin and heparan sulphate showed a much higher inhibitory potency towards the plasma membrane ganglioside GM3 sialidase than towards the lysosomal onw.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential uPAR recruitment in caveolar‐lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid‐rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar‐lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro‐angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid‐supported mobile bilayer lipid membranes with raft‐like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR‐GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1‐enriched biomimetic membranes, were validated by identifying a pro‐angiogenic activity of GM1‐enriched EPCs, based on GM1‐dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti‐angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar‐raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar‐raft partitioning of uPAR, as opposed to control and GM3‐challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.     

The N-acetylgalactosamine residue of exogenous GM2 ganglioside is recycled for glycoconjugate biosynthesis in rat liver.

A metabolic recycling of N-acetylgalactosamine (GalNAc), liberated from exogenous GM2 ganglioside [nomenclature of Svennerholm (1964) J. Lipid Res. 5, 145-155; IUPAC-IUB recommendations (1977) Lipids 12, 455-468], is demonstrated in rat liver. After the injection of a GM2 ganglioside isotopically radiolabelled on the terminal GalNAc residue ([GalNAc-3H]GM2), the liver retained a large amount of radioactivity distributed among: (1) a glycoprotein/glycosaminoglycan fraction, (2) a ganglioside fraction; and (3) a free-sugar fraction. Furthermore, volatile radioactivity was also found. The relative incorporation in the above fractions was time-dependent. The glycoprotein/glycosaminoglycan fraction contained radioactivity that was located on the GalNAc and GlcNAc residues. The ganglioside fraction was composed of two main families: gangliosides formed by a recycling of the liberated GalNAc, and gangliosides derived by direct utilization of the administered GM2. The free-sugar fraction contained mainly GalNAc. We suggest that GalNAc, after being released in the course of intra-lysosomal ganglioside catabolism, crosses the lysosomal membrane and passes into the cytosol, where the part not degraded is re-utilized for the biosynthesis of the different glycoconjugate classes.     

Lysosomal membrane cholesterol dynamics

Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, the assumption that cholesterol transfers out of the lysosome by rapid (minutes), spontaneous diffusion has heretofore not been tested. As shown herein, lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol-mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane and other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosomes and purified lysosomal membranes was extremely slow, t(1/2) >4 days. This was >100-fold longer than that reported in intact cells (2 min) and 40-60-fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of sterol transfer was maximally increased nearly 80-fold and the organization of cholesterol in the lysosomal membrane was rapidly altered. Nearly half of the essentially nonexchangeable sterol in the lysosomal membrane was converted to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lysosomal membrane was extremely slow, inconsistent with rapid spontaneous diffusion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consistent with cholesterol leaving the lysosome earlier in the endocytic process and/or with cholesterol transfer out of the lysosome being mediated by additional process(es) extrinsic to the lysosome and lysosomal membrane.     

Isolation and characterization of epididymal epithelial cell plasma membranes

Sperm maturation and storage occur in a unique milieu created in large part by the epididymal epithelium. To learn more about the interaction of the epididymal epithelial cell with both luminal and systemic environments, we now report on the preparation and characterization of epididymal epithelial cell plasma membranes. A preparation enriched for epididymal epithelial cell plasma membranes was isolated from collagenase-digested epididymal tubule fragments by hand-Dounce homogenization, differential centrifugation, and sucrose gradient centrifugation. The final membrane fraction was enriched 11-fold for the plasma membrane marker 5'-nucleotidase; 2.6-fold for the lysosomal marker acid phosphatase, and 3-fold for the Golgi marker thiamine pyrophosphatase. No enrichment was observed for mitochondrial or endoplasmic reticulum enzyme markers. Specific and saturable transferrin-binding activity was also detected in the final preparation. Electron microscopy revealed the presence of vesicles and sheets of membranes as well as an occasional Golgi apparatus. The plasma membrane fraction was used to generate monoclonal antibodies. Of 102 wells exhibiting growth, 12 were positive by immunofluorescent staining of frozen sections. Ten of these recognized determinants in epithelial cells, and 2 stained peritubular smooth muscle cells. Most of the epithelial cell-specific antibodies stained brush border alone or in combination with the basolateral plasma membrane. Three antibodies stained the Golgi apparatus. Most antibodies were specific for particular epididymal regions, 3 also recognized determinants in the kidney, and 1 stained residual bodies in the testis.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Occurrence in Brain Lysosomes of a Sialidase Active on Ganglioside

A lysosomal preparation, obtained from brain homogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40-120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GD1a and fluorimetrically with 4-methylumbelliferyl-1-alpha-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma membrane-bound enzyme. The apparent Km values for GD1a and MUB-NeuAc were 1.5 X 10(-5) and 4.2 X 10(-5) M, respectively, for the lysosomal enzyme and 2.7 X 10(-4) and 6.3 X 10(-5) M for the plasma membrane-bound one. Triton X-100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of Plasma Membrane of Guinea Pig Peritoneal Macrophages

A method using sucrose density gradient centrifugation is described for the purification of plasma membrane of guinea pig peritoneal exudate macrophages. Assays for composition and for marker enzyme activities have been modified for use with the small amounts of subcellular macrophage material. The plasma membrane was obtained in 57% yield and contained 7% of the protein. The purified plasma membrane fraction is five-fold enriched in phospholipid to protein ratio and contains no contaminating DNA, none of the cytoplasmic marker lactate dehydrogenase, no detectable mitochondrial contamination and a low contamination with lysosomal enzymes (7%). Purified plasma membrane containing 4 mg of protein can be prepared from 1 ml of pelleted macrophages in a one-day operation.