Library

Notes: Abstract: The total pellet from pig forebrain (from which the cytosolic sialidase was completely washed out) was treated with phosphatidylinositol phospholipase C (PIPLC) and centrifuged at high speed. The supernatant contained sialidase and 5′-nucleotidase activities. The greatest liberation of sialidase was obtained after incubation for 20 min with PIPLC at 37°C using pH 6.0 and a ratio between PIPLC (as units) and protein of 1.6. Under these conditions, the release of sialidase, 5′-nucleotidase, and protein was 22, 50, and 18.5%, respectively. On treatment with PIPLC, a purified preparation of pig brain neuronal (synaptosomal) membranes released 28% of its sialidase, whereas a purified preparation of pig brain lysosomes did not liberate any sialidase activity. The pH optimum of sialidase present in the supernatant obtained after PIPLC treatment of the total pellet was 4.2, the same as that of the enzyme embedded in the membrane. When this supernatant was subjected to ammonium sulfate fractionation, 88% of its sialidase, having a pH optimum of 4.2, was recovered in the fraction precipitated between 20 and 45% of salt saturation and subsequently dialyzed. Ammonium sulfate treatment caused the appearance of a second sialidase activity, having a pH optimum of 6.6 and behaving on fractionation similarly to the pH 4.2 sialidase. The Km and Vmax values of pH 4.2 and pH 6.6 sialidase were similar (1.48 ± 10−-4 and 0.98 ± 10−-4M for Km and 1.6 and 1.4 mU/mg of protein for Vmax, respectively), whereas the stability on standing at 4°C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase. Both enzyme activities could be separated by Sepharose 6B column filtration in a unique fraction that was eluted between catalase and bovine serum albumin. These results suggest that (a) the plasma membrane fraction from pig forebrain contains two sialidases (pH 4.2 and 6.6) that are presumably linked via a glycosylphosphatidylinositol anchor, sensitive to PIPLC action, and (b) pH 6.6 sialidase may be in the membrane under the inhibition of a factor that is liberated by PIPLC treatment and removed during ammonium sulfate fractionation and subsequent dialysis.

Notes: Abstract: Five major gangliosides (GM1, GD1a, GDlb, GTlb, and GQlb) were extracted and isolated by normal-phase HPLC from the forebrain of Sprague-Dawley rats of ages ranging from 3 days to 24 months. Each ganglioside was fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base moiety. At all ages, the C18:1 and C20:l long-chain base species predominated, whereas the C18:0 and C20:0 ones represented 1–3% of the total. The C18:l long-chain base species, predominant at 3 days (91–96%), diminished with age and reached, at 2 years, 73%, 65%, 61%, 59%, and 45% of the total for GDla, GM1, GTlb, GDlb, and GQlb, respectively. The content of the C20:1 long-chain base species, low at birth (4–9%), increased with age in all gangliosides and reached, at 2 years, 27–55% of the total. The developmental behavior of the ganglioside species containing the C18:1 long-chain base was characterized by the following: (a) a biphasic profile with a maximum around 15 days for GD1a, the most abundant ganglioside at all ages; (b) an increase until 6 months for GM1; (c) a sharp decrease until 30 days, followed by leveling for GTlb; and (d) a low, constant level for GDlb and GQlb. All the ganglioside species containing the C20:1 long-chain base showed a constant increase during development, the increase being more marked in the first 30 days. The molecular species of all gangliosides carrying the C18:1 long-chain base were virtually devoid of 20:0 fatty acids and carried a higher content of 18:0 fatty acids than the corresponding C20:l long-chain base species (average 80 versus 57%). Moreover, in the C18:1 long-chain base species, the 18:0 fatty acid content diminished with age from 89 to 72%, with a concurrent increase of 16:0 and 18:1 fatty acids, whereas the C20:l long-chain base species had an age-constant fatty acid composition.

Notes: GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long- chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a(∼9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long- chain-base analysis showed that newly-formed GM1 contained ∼68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing ∼ 10% hydrolysis. Long- chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipal-mitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base.

Notes: The binding of GM1, ganglioside to crude preparations of rat brain neuronal membranes was studied, the following results being obtained: (a) the binding process followed a biphasic kinetics, which displayed a break at 0.07–0.08 X 10−6m GM1, concentration; (b) the features of the binding process at GM1, concentrations below the break and, over the break, above 10-6m appeared to be different. Below the break the process proceeded slowly and brought a stable and irreversible association of GM1, molecules to the membranes. Over 10-6m the process was much more rapid and caused GM1, molecules to interact in such a way that they were releasable by washing and could exchange with newly added free ganglioside; (c) the two binding processes displayed the characteristics of a saturation phenomenon; (d) in both cases, GM1, taken up was freely available to galactose oxidase, indicating that the oligosaccharide chains protrude from the membrane surface. We postulate that GM1, occurs, below and above the break, in different physical forms, each of them having a different mechanism of interaction with the membrane. Above 10-6m GM1, interacts as micelles, and the basis of the micelle-membrane inter action is a fusion process. Below the break, in the 10−8–10−7m range, the binding is the result of hydrophobic interactions between sites on the membrane and the hydrophobic portion of individual ganglioside molecules, most likely in the monomeric form. Toffano G. et al. Interactions of GM1, ganglioside with crude rat brain neuronal membranes. J. Neurochem.35, 861–866 (1980).

Notes: —The activity of brain membrane-bound neuraminidase on endogenous and exogenous substrates was comparatively studied in various animals (rat, chicken, rabbit, pig, calf and human). The maximum rate of hydrolysis of endogenous substrates by membrane-bound neuraminidase (using a crude preparation of the enzyme) was different in the various animals (from 0·05 to 0·73 units, referred to 1 mg protein) and was obtained under similar but not identical optimum conditions (pH from 4·1 to 5·1; requirement or not of Triton X-100). The maximum degree of hydrolysis of endogenous substates was also different (from 15 to 27 nmol released NeuNAc/mg protein) and was obtained within different incubation periods (from 2 to 18 h). It corresponded (in rabbit, calf, human brain only), or not, to the actual exhaustion of the endogenous substrates.The endogenous substrates were recognized as both gangliosides and sialoglycoproteins. The extent of hydrolysis of sialoglycoproteins varied from 1·5% in rabbit to 15·6% in chicken brain; the hydrolysis of gangliosides (ranging from 14·1% in pig to 53·7% in rabbit brain) reached only in some animals (rabbit, calf, human) the complete transformation of major oligosialogangliosides into the neuraminidase resistant monosialoganglioside GMI. Upon addition of exogenous substrates (sialyl-lactose, ganglioside GD1a, brain sialopeptides, ovine submaxillary mucin) the actual rate of liberation of total NeuNAc (from both endogenous and exogenous substrates) considerably exceeded, although at a different extent (depending on the animal and on the added substrate used) the rate of hydrolysis of sole endogenous substrates. The possibility of an accurate assay of brain membrane-bound neuraminidase in a crude enzyme preparation is evaluated and guidelines for the assay procedure suggested.

Notes: Abstract— Two new gangliosides were isolated in pure form from beef brain. They were provisionally named ganglioside G5a and G5b. Ganglioside GSa is a monosialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal (1 —〉 3) GalNac (1—〉 4) Gal (1 —〉 4) Glc (1—〉) ceramide, most likely with β-linkages. Fucose is linked to the 2-position of external galactose, N-acetylneuraminic acid to the 3- position of internal galactose. Ganglioside G5b is a mixture of at least two isomeric disialogangliosides containing N-acetylneuraminic acid and N-glycolylneura-minic acid. The major isomer has the following structure: NeuNac (α,2—〉3) Gal (β,1—〉3) GalNac (β, 1 —〉 4) (NeuNglα, 2 —〉 3) gal (β,1—〉4) Glc (β,1 —〉)-ceramide. The minor isomer contains N-acetylneur-aminic acid and N-glycolylneuraminic acid in an inverted linkage position.

Notes: A new ganglioside, provisionally named GLIVa, was isolated in pure form from pig cerebellum. Ganglioside GLIVa is a disialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal, β 1 → 3 GalNAc, β 1 → 4 Gal, β 1 → 4 Glc, β 1 → Cer.Fucose is α-glycosidically linked to the 2-position of external galactose and one N-acetylneuraminic acid is linked to the other one by an α, 2 → 8 linkage. Thus the total structure of ganglioside GLIVa is the following: Fuc, α 1 → 2 Gal, β 1 → 3 GalNAc, β 1 → 4 (NeuAc, α 2 48 NeuAc, α 2 → 3) Gal, β 1 → 4 Glc, β 1 → Ceramide. According to the IUPAC-IUB Commission on Biochemical Nomenclature is indicated as II3α(NeuAc)2 IV2αFuc-GgOse4Cer.