Sergio Marchesini

A new procedure for the extraction, purification and fractionation of brain gangliosides

Abstract 1. 1. A procedure for the extraction, separation and purification of brain gangliosides is described, which involves: exhaustive extraction of brain with buffered tetrahydrofuran; partition of the extract first with ethyl ether, then with distilled water; dialysis of the obtained aqueous phase, and chromatography of the dialyzed solution on silica gel column. The residue after exhaustive extraction contains all brain glycoproteins. 2. 2. This procedure, compared with the conventional chloroform-methanol procedure, was proved to achieve a more complete extraction of gangliosides with no concurrent solubilization of glycoproteins and to guarantee the absence of any significant degradation. Thus, the present procedure is able to provide a final preparation of gangliosides free of glycoproteins, and a preparation of glycoproteins free of gangliosides.

CYTOSOLIC GANGLIOSIDES: OCCURRENCE IN CALF BRAIN AS GANGLIOSIDE-PROTEIN COMPLEXES

—Calf brain was treated in order to prepare separately the cytosol from neuronal bodies and glial cells, and the cytosol from nerve endings. The first cytosol contained 29 μg of ganglioside bound sialic acid/g fresh tissue, the latter 3.1 μg. Upon addition of ammonium sulphate until saturation the gangliosides contained in the two cytosols precipitated and were totally recovered in the pellet. while, under the same conditions, pure gangliosides were completely soluble. After stepwise ammonium sulphate fractionation all the different fractions obtained contained gangliosides and carried an approximately constant ganglioside/protein ratio. Thus cytosolic gangliosides occur in calf brain as ganglioside‐protein complexes.

Influence of age and sex on five human plasma lysosomal enzymes assayed by automated procedures

Automated fluorimetric procedures for the assay of five lysosomal glycohydrolases-beta-N-acetylglucosaminidase; beta-galactosidase; beta-glucuronidase; alpha-mannosidase; alpha-fucosidase-in human plasma were set up. A Carlo Erba autoanalyser CLA 1500, provided with a sampler refrigerating unit and connected with a recording Turner Mod 111 fluorimeter was employed. The automated procedures, under the established optimal conditions, proved to be highly accurate and reproducible. Using the automated assay procedures the effect of sex and age on the plasma levels of the same enzymes was studied. 1273 randomly selected health subjects were studied. No sex differences were observed for all the enzymes studied with the exception of beta-glucuronidase which displayed higher values (about 30%) in males from 25 to 60 years. The developmental profiles of all enzymes in females and males were similar and characterised by: (a) absolute maximum level in the umbilical cord blood; (b) absolute minimum level at 10-14 years; (c) decrease to a second minimum occurring around 35 years (not displayed by beta-galactosidase and by beta-glucuronidase in males); (e) slow further increase up to the elderly level which was then maintained till the oldest age examined, 74 years.

N-Pyrene dodecanoyl sulfatide as membrane probe: a study of glycolipid dynamic behavior in model membranes

An N-linked pyrene-dodecanoyl sulfatide was employed to measure the ratio of excimer fluorescence to monomer fluorescence intensities (E/M). The E/M values provided information about both the dynamic behavior and the structural distribution of the labelled glycolipid in note dispersion of micellar sulfatides and multilamellar vesicles of different phospholipids. Most of the labelled sulfatide seems to be located in domains sequestered from the surrounding phospholipids still above the phase transition temperature of the vesicles. The glycolipids sequestered in these domain environments are less sensitive to the structural changes that the addition of cholesterol or Ca2+ can induce in the phospholipid regions during the phase transition.

Fluorospectroscopic studies of mixtures of distearoylphosphatidylcholine and sulfatides with defined fatty acid compositions

Simple study models characteristic for lamellar organization of distearoylphosphatidylcholine and sulfatide have been prepared for fluorospectroscopic investigations on the influence of these glycolipids on the chemico-physical properties of lecithin bilayers. The motion of 1,6-diphenyl-1,3,5-hexatriene in mixed lecithin-sulfatide bilayers changed with temperature, with the compositional ratio of the two lipids, with the presence of divalent cations such as Ca2+ and with the fatty acid composition of sulfatide moiety. Steady-state fluorescence measurements of the average motion of the fluorophore permit evaluation of the gel to liquid-crystalline phase transition in all these membrane models containing different sulfatides.