Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes.

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 x 10(4). On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 x 10(5)) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 X 10(5), and a ganglioside/protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides GD1a and GD1b, which, like GT1b, are potential substrates for the enzyme and GM1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. GT1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with GD1a and GD1b.

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes.

The interactions of ganglioside GM1 with human and fetal calf sera were studied, the following main results being obtained: (a) GM1, upon incubation with both sera gave origin to two GM1-protein complexes, which also occurred after interaction of GM1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore GM1 appeared to specifically bind serum albumin and to form GM1-albumin complexes. (b) GM1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10(-6) M), was time and concentration dependent, and resulted in a relevant degree of GM1 complexation (up to 80% of total GM1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 . 10(-4) M GM1, the ratio between bound and total GM1 increased linearly with increasing GM1 concentration; in the second phase, occurring above 2 . 10(-4) M, the ratio remained practically constant. After these findings it should be expected that GM1, when present in serum containing systems, forms complexes with albumin. This should be approximately considered when studying the effects of exogeneous GM1 in in vivo and in vitro (tissue cultures) systems.

Cytosolic sialidase from pig brain: a 'protein complex' containing catalytic and protective units.

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved into a catalytic unit (M(r) 30 kDa), active but very liable upon storage at 4 degrees C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2 x 10(-5) and 1.6 x 10(-5) M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any beta-D-galactosidase, beta-D-N- acetylglucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxypeptidase activities.

Occurrence of sialidase activity in two distinct and highly homogeneous populations of lysosomes prepared from the brain of developing mouse.

Light and heavy lysosomes of mouse forebrain were separated from each other by centrifugation on a Percoll gradient. Light lysosomes were then freed from mitochondria and membranes by sucrose density gradient centrifugation and further purified by floatation-centrifugation on a sucrose gradient. The final preparations of light and heavy lysosomes, fairly homogenous, carried sialidase activity, assayed on MU-NeuAc. The optimal pH was 4.0 and 4.2, the apparent Km value 2.8 x 10(-5) M and 4.2 x 10(-5) M and the apparent Vmax value 0.11 and 0.47 mU.mg-1 protein, for the light and heavy lysosome sialidase, respectively. From 4 days to adulthood the specific activity of the light and heavy lysosome sialidase increased 3-fold and 1.7-fold, respectively.

Interactions of pig brain cytosolic sialidase with gangliosides. The formation of catalytically inactive enzyme-ganglioside complexes requires homogeneous ganglioside micelles and is a reversible phenomenon.

Cytosolic sialidase A, obtained from pig brain and purified, interacts with ganglioside GT1b giving two catalytically inactive enzyme-ganglioside complexes. Treatment of these complexes with Triton X-100 under given conditions (1% detergent; 1 h at 37 degrees C; 0.1 M acetic acid-sodium acetate buffer, pH 4.8) leads to the liberation of part of the enzyme (about 47%) in a free and fully active form. Reversible inactivation of cytosolic sialidase requires the presence of homogeneous micelles of GT1b or of mixed micelles (for instance Triton X-100 and GT1b) with a high GT1b content. Triton X-100/ganglioside mixed micelles with a molar ratio above 50, as well as small unilamellar vesicles of egg yolk lecithin and GT1b (7-15 mol%), did not inactivate the enzyme at all; on the contrary these forms of ganglioside dispersion behaved as excellent substrates for the enzyme. It is to be concluded that under in vitro conditions the ability of ganglioside to interact with cytosolic sialidase, giving rise to catalytically inactive complexes or to Michaelis-Menten enzyme-substrate complexes, depends on the supramolecular organization of the ganglioside molecules. Arrangements of tightly packed molecules with strong side-side interactions facilitate the formation of complexes with the enzyme; arrangement with separated and loosely interacting molecules facilitates binding at the catalytically active site of the enzyme.

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-(3)H]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-(3)H]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.

Cytosolic sialidase in developing rat forebrain.

The nature and developmental profile of the soluble sialidase of rat forebrain were studied from birth to 150 days. Forebrain was extracted by two procedures, one (mild) preserving, the other (drastic) destroying nerve endings. The soluble extracts obtained by the mild procedure contained 64-78% of the total tissue cytosol, assayed as lactate-dehydrogenase; those obtained by the drastic procedure 87-94%. These latter extracts were considered as the soluble fraction containing 'all' tissue cytosol. The cytosolic origin of the sialidase contained in the soluble extracts at all examined ages was suggested by the following evidence: (a) during extraction sialidase behaved as lactate-dehydrogenase and quite differently from ?-hexosaminidase and ?-galactosidase, enzymes of lysosomal nature present in the same extracts, (b) the sialidase content of the extract was not influenced by the presence or absence of EDTA in the medium, (c) the sialidase content in the extracts did not diminish even after prolonged centrifugation (2 h) at high speed (150,000 g). The content of cytosolic sialidase referred to g fresh tissue increased from birth to 20 days, and slowly decreased thereafter. Till 20 days the content and the developmental trend of the cytosolic enzyme were similar to that of the better known membrane bound sialidase. This latter enzyme, however, reached its maximum at about 60 days of age. The specific activity of the cytosolic sialidase was lower till 10 days of age, higher from 10 to 30 days, and equalled that of the membrane bound enzyme during adult life. Therefore rat forebrain cytosolic and membrane bound sialidases, also from the developmental point of view, behave as different enzymes.

Cytosolic sialidase from the nerve endings of developing rat forebrain.

The developmental profile of cytosolic sialidase from the nerve ending and the nerve ending-free compartments of rat forebrain was studied from birth to 150 days. Soluble extracts containing the cytosol from nerve endings and nerve ending-free tissue were separately prepared; the recovery of cytosol in the two extracts was followed by assaying lactate-dehydrogenase. In both cytosolic extracts the content of sialidase and lactate-dehydrogenase had a marked and progressive enhancement from the 5th to the 20th day of life and then maintained a constant level through adult life. However the rate of lactate-dehydrogenase and sialidase increase in the two cytosolic compartments was different. Lactate-dehydrogenase increased at a very similar rate in the two cytosols. Instead the rate of sialidase increase was greater in the cytosol from nerve endings from the 5th to the 10th day of life, and, inversely, in the cytosol from nerve ending-free tissue from 10 to 20 days. Between the 5th and 10th day of life the nerve ending cytosol underwent an enrichment of sialidase which was several times higher than that of lactate-dehydrogenase; in the other periods of forebrain development sialidase and lactatedehydrogenase moved in parallel. The variations with age of cytosolic sialidase from the nerve ending-free tissue seemed to follow the overall process of brain development. The nerve ending soluble sialidase would more specifically reflect formation of synaptic junctions in some brain regions in a well defined period of brain maturation.

Studies on peroxidation processes of model membranes and synaptosomes: role of phosphatidic acid.

Dipalmitoylphosphatidic acid (DPPA) was found to exert a strong inhibitory effect on Fe-induced peroxidation of arachidonic acid inserted into liposomal dipalmitoylphosphatidylcholine (DPPC) vesicles. This inhibition was quite effective both below and above the phase transition temperature of the liposomes. Moreover, we demonstrated the antiperoxidative activity of phosphatidic acid (PA) in synaptosomal membranes. PA enriched synaptosomes were prepared by the stimulation of the endogenous phospholipase D activity or by the incubation of the synaptosomes with Streptomyces chromofuscus phospholipase D. The possible contribution of PA to the in vivo defense mechanism against free radical-induced damage is discussed.

Approaches in the study of ganglioside metabolism.

Ganglioside GM1, 3H-labeled in the sphingosine or terminal galactose moiety was injected into mice and its metabolic fate in the liver was followed. After administration of sphingosine-labeled GM1 all major liver gangliosides (GM3, GM2, GM1, GD1a-NeuAc, NeuG1) became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest on GM1, followed by GM2, GM3 and GD1a-NeuAc, NeuG1. Several neutral glycosphingolipids and sphingomyelin were also formed. After administration of galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-NeuAc, NeuG1, both carrying the radioactivity on the terminal galactose residue, with no formation of labelled neutral glycosphingolipids. Subcellular studies gave clear evidence that GM1, after being taken up by the liver, was mainly degraded to GM2, GM3 and neutral glycosphingolipids at the level of lysosomes. A part of it was sialylated to more complex gangliosides and some of its metabolic by-products were used for the biosynthesis of other sphingolipid species, likely at the level of the Golgi apparatus. All this suggests that exogenous GM1 is introduced in the metabolic routes of endogenous gangliosides and of other sphingolipids, which are operating in the liver.

Lesser proximal interphalangeal joint arthrodesis: a retrospective analysis of the peg-in-hole and end-to-end procedures.

A retrospective study was performed to compare the prevalence of complications in peg-in-hole and end-to-end arthrodesis procedures. The authors reviewed 177 second, third, and fourth proximal interphalangeal joint fusions for the correction of hammer toe deformities in 85 patients from 1988 to 1998 at the Temple University School of Podiatric Medicine. The average age of the patients was 49 years. Sixteen percent (14) of the subjects were male and 84% were (71) female. Upon follow-up, the fourth digit was generally associated with a greater number of complications for the end-to-end and peg-in-hole procedures, with the second digit being the most common site of fusion. The prevalence of complications was evaluated using contingency table analysis and expressed as a percent of total complications (27%, the end-to-end group; 17%, the peg-in-hole group). A subset of complications deemed clinically relevant was also computed. Similarly, the prevalence of clinically relevant complications for the end-to-end (10%) and the peg-in-hole (9%) procedures was not statistically significant. Therefore, this study showed no statistically significant differences in the total or clinically relevant complications between end-to-end and the peg-in-hole arthrodesis procedures.

Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide beta 1----4galactosyltransferase.

The presence and subcellular localization of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) was investigated in rat liver. For this purpose, purified Golgi apparatus, endoplasmic reticulum, and plasma membrane fractions were prepared from the liver and used as the enzyme source for detecting GalT-2. A pure Golgi apparatus, highly enriched in many glycosyltransferases, was the only fraction where GalT-2 was measurable. The reaction product formation rate under appropriate assay conditions, which requires high detergent concentration and Mn2+, was low but comparable with that of other glycosyltransferases. The product formation was stimulated by exogenously added acceptor GlcCer, donor UDP-Gal, and Golgi protein. The reaction product was a single spot that was identified by chromatographic behavior, sensitivity to beta-galactosidase, and permethylation studies as Gal beta 1----4Glc beta 1----1'Cer (lactosylceramide). A metabolic experiment, performed by determining the glycosphingolipids which became radioactive in the above subcellular fractions prepared from the liver of animals treated with glucose-labeled glucosylceramide, further indicated that the in vivo glycosylation of glucosylceramide takes place in the Golgi apparatus.

Age-related differences in synaptosomal peroxidative damage and membrane properties.

Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+, K(+)-ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age-related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age-dependent decrease in membrane fluidity, a reduction of Na+, K(+)-ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K(+)-ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to ageing processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.

Presence in human erythrocyte membranes of a novel form of sialidase acting optimally at neutral pH.

The feature of intact human erythrocytes and erythrocyte white ghosts is a unique sialidase activity with acidic optimal pH (acidic sialidase). The treatment of white ghosts with mildly alkaline isotonic solutions at 37 degrees C, like that used to produce resealed ghosts, is accompanied by the expression, together with the acidic sialidase, of a novel sialidase with a pH optimum of 7.2 (neutral sialidase) that remained masked in the inside-out vesicles prepared from white ghosts. Exhaustive treatment of resealed ghosts with Bacillus Thuringiensis phosphatidylinositol-specific phospholipase C causes an almost complete release of the acidic sialidase, with the neutral enzyme remaining totally unaffected. The treatment of resealed ghosts with 1.2% Triton X-100 resulted in the solubilization of only the neutral sialidase, whereas 3.6% octylglucoside also solubilized the acidic sialidase. The neutral enzyme affected not only the artificial substrate but also any sialoderivatives of a ganglioside, glycoprotein, and oligosaccharide nature; the acidic enzyme did not affect sialoglycoproteins. Erythrocyte endogenous gangliosides were hydrolyzed by both sialidases, whereas the endogenous sialoglycoproteins responded to only the neutral enzyme. It was definitely proved that the acidic sialidase is located on the outer erythrocyte membrane surface, so presumably the neutral enzyme has the same location. It could be that the newly discovered neutral sialidase has a physiologic role in the releasing of sialic acid from erythrocytes during the erythrocyte aging process, leading to eventual phagocytosis by macrophages.

Different behavior of ghost-linked acidic and neutral sialidases during human erythrocyte ageing.

Acidic and neutral sialidases (pH optimum 4.7 and 7.2, respectively) were assayed on human circulating erythrocytes during ageing. The assays were performed on intact erythrocytes and resealed erythrocyte ghost membranes. From young to senescent erythrocytes the acidic sialidase featured a 2.7-fold and 2.5-fold decrease in specific activity when measured on intact cells or resealed ghost membranes, whereas the neutral sialidase a 5-fold and 7-fold increase, respectively. The Ca2+-loading procedure was employed to mimic the vesiculation process occurring during erythrocyte ageing. Under these conditions the released vesicles displayed an elevated content of acidic sialidase, almost completely linked through a glycan phosphoinositide (GPI) anchor but no neutral sialidase activity, that was completely retained by remnant erythrocytes together with almost all the starting content of sialoglycoconjugates. The loss with vesiculation of acidic sialidase with a concomitant relative increase of neutral sialidase was more marked in young than senescent erythrocytes. The data presented suggest that during ageing erythrocytes loose acidic sialidase, and get enriched in the neutral enzyme, the vesiculation process, possibly involving GPI-anchors-rich membrane microdomains, being likely responsible for these changes. The enhanced neutral sialidase activity might account for the sialic acid loss occurring during erythrocyte ageing.

Interactions of proteins with ganglioside-enriched microdomains on the membrane: the lateral phase separation of molecular species of GD1a ganglioside, having homogeneous long-chain base composition, is recognized by Vibrio cholerae sialidase.

The thermotropic behavior (studied by high-sensitivity differential scanning calorimetry) and susceptibility to Vibrio cholerae sialidase hydrolysis of large unilamellar vesicles of dipalmitoyl-phosphatidylcholine, containing native GD1a ganglioside or the molecular species of GD1a containing C18:1 or C20:1 long-chain base (C18:1 GD1a; C20:1 GD1a), were studied. Vesicles containing ganglioside (10% in molar terms) showed the presence in the heat capacity function of a second minor peak besides the phospholipid main transition peak. The presence of a second peak is much more evident with C20:1 GD1a than with C18:1 GD1a, the difference being potentiated by Ca2+ and indicating a different tendency of the CD1a molecular species to undergo lateral phase separation. The scans of vesicles containing native GD1a showed the features of those obtained with C18:1 GD1a and C20:1 GD1a, indicating that the main components of native GD1a, C18:1 GD1a and C20:1 GD1a, maintain their individual aggregative properties. V. cholerae sialidase affects vesicle-bound GD1a at a much higher rate (17-25-fold) than it does micellar GD1a, the activation by Ca2+ being 3- and 2-fold, respectively. The Vmax values were identical on C18:1 GD1a and C20:1 GD1a in micellar dispersions, whereas they were markedly higher (from 20 to 50%) on C18:1 GD1a than on C20:1 GD1a in vesicular dispersions. Exhaustive sialidase hydrolysis of vesicles carrying native GD1a produced C18:1 GM1 and C20:1 GM1 in the same proportion as the C18:1 and C20:1 species present in native GD1a (53.9% and 46.1%).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic gangliosides of rat brain: their fractionation into protein-bound complexes of different ganglioside compositions.

The sialic acid moiety of rat brain cytosolic gangliosides was radiolabeled by intracranial injection of N-(3H)acetylmannosamine. Upon ammonium sulphate fractionation, Sepharose 6B gel filtration, and hydroxylapatite-cellulose chromatography, ganglioside-bound radioactivity of brain cytosolic extract followed the behavior of protein and not that of purified gangliosides. This indicates that cytosolic gangliosides occur as ganglioside-protein complexes. By application of hydroxylapatite-cellulose column chromatography, fractions were obtained having different ganglioside composition. In particular, one fraction contained GM1, one GD1a, and one GT1b with a ganglioside homogeneity better than 95% in each fraction. This indicates the occurrence in brain cytosol of a GM1-protein complex, a GD1a-protein complex, and a GT1b-protein complex.