V. Zambotti

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.

Parallelism of subcellular location of major particulate neuraminidase and gangliosides in rabbit brain cortex

Abstract The subcellular distribution of major particulate neuraminidase and gangliosides in the rabbit cortex was determined. The neuraminidase and gangliosides were found to be present in: (a) the nerve endings; (b) the light membranes of microsomal origin ; (c) the myelin-rich preparation obtained from the crude mitochondrial fraction. Their distribution patterns were very similar also from the quantitative point of view; in fact 40 % of the neuraminidase activity and 43.5 % of the gangliosides were recovered in the nerve endings; 50% of the enzyme and 45% of the gangliosides in the light microsomal membranes; 8 % of the neuraminidase and 11 % of the gangliosides in the myelin-rich preparation. Conversely, the preparations enriched in nuclei, mitochondria and lysosomes were practically devoid of both neuraminidase and gangliosides. The ganglioside patterns of the different subcellular fractions were similar, except for the myelin-rich subfraction which contained higher amounts of monosialoganglioside GM1. The microsomal light membranes had a slightly lower content of trisialoganglioside GTIb and tetrasialoganglioside GQ1 than the nerve endings. These results may be considered consistent with the hypothesis that neuraminidase and gangliosides are fundamental components of the neuronal plasma membrane, thus following the distribution of the different neuronal fragments after homogenization and fractionation.

Studies on brain cytosol neuraminidase: I. Isolation and partial characterization of two forms of the enzyme from pig brain

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.

MEMBRANE-BOUND NEURAMINIDASE IN THE BRAIN OF DIFFERENT ANIMALS: BEHAVIOUR OF THE ENZYME ON ENDOGENOUS SIALO DERIVATIVES AND RATIONALE FOR ITS ASSAY

—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.

Glycolipids, Glycoproteins, and Mucopolysaccharides of the Nervous System

What can We Expect from the Study of Neurological Mutants.- Section I Chemistry and Metabolism of Glycoproteins and Mucopolysaccharides of the Nervous System.- Chemistry and Metabolism of Glycopeptides Derived from Brain Glycoproteins.- Chemistry and Metabolism of Glycosaminoglycans of the Nervous System.- Biosynthesis of Brain Glycoproteins.- Glycoproteins of the Synaptosomal Plasma Membrane.- Glycoproteins During the Development of the Rat Brain.- Section II Chemistry and Metabolism of Glycolipids of the Nervous System.- Recent Advances on the Chemistry and Localisation of Brain Gangliosides and Related Glycosphingolipids.- Enzymatic Aspects of Sphingolipid Metabolism.- Recent Studies on the Enzymes that Sinthesize Brain Gangliosides.- Brain Neuraminidases.- Influence of Thyroid upon Lipid Content in Developing Cerebral Cortex and Cerebellum of the Rat.- Section III Separation and Purification of the Subcellular Components and Plasma Membranes of the Nervous System.- Methods of Separating the Subcellular Components of Brain Tissue.- The Isolation and Characterization of Synaptosomal Plasma Membranes.- Section IV Chemical Pathology and Diagnosis of Lipid and Mucopolysaccharide Storage Diseases.- Glycolipid, Mucopolysaccharide and Carbohydrate Distribution in Tissues, Plasma and Urine from Glycolipidoses and Other Disorders.- Neuropathology of Glycopeptides Derived from Brain Glycoproteins.- Effect of Bacterial Neuraminidase on the Isoenzymes of Acid Hydrolases of Human Brain and Liver.- Problems in the Chemical Diagnosis of Glycoprotein Storage Diseases.- Progress and Problems on Fucosidosis and Mucolipidosis.- Early Diagnosis of Glycolipidosis.- Patterns of Brain Ganglioside Fatty Acids in Sphingolipidoses.

Assay of brain particulate neuraminidase III. Preparation of the enzyme devoid of endogenous substrates

Abstract 1. 1.|The crude preparation of brain particulate neuraminidase (mucopolysaccharide N-acetylneuraminylhydrolase, EC 3.2.1.18) (0–105 000 × g pellet, prepared from rabbit brain homogenate in isotonic sucrose) was depleted from endogenous (intrinsic) substrates by autolysis performed under the following conditions: 0.18 M sodium acetate buffer, 0.3% Triton X-100, final pH 4.2, 25°, 5 h incubation. 2. 2.|After this treatment only 54% protein, 51% sialoglycoproteins, 89% gangliosides, 75% total and lipid-bound phosphorus remained particulate. Neuraminidase remained firmly particulate and fully active (97% recovery). Thus, the 105 000 × g sediment obtained after the above treatment is the enzyme preparation devoid of endogenous substrates. 3. 3.|The basic properties of the enzyme present in the preparation devoid of endogenous substrates were the same observed in the starting crude preparation. The Km for disialoganglioside GD1a was 4.8·10−6 M; the vmax 1.6 nmoles released N-acetylneuraminic acid per min. 4. 4.|The examined properties of particulate neuraminidase depleted from endogenous substrates by our procedure were very similar to those of the enzyme prepared according to Z. Leibovitz and S. Gatt (Biochim. Biophys. Acta, 152 (1968) 136). However, the final enzyme recovery was 5 times higher with our procedure.

Studies of brain membrane-bound neuraminidase. II. Effect of detergents on the kinetics of the enzyme prepared from calf brain

Abstract 1. 1. The influence of various detergents on the kinetics of membrane-bound neuraminidase acting on gangliosides (amphipatic substances) was studied. The enzyme was prepared from calf brain and disialoganglioside GD1a was used as substrate. 2. 2. In the absence of detergents the enzymatic hydrolysis of GD1a follows hyperbolic kinetics with a maximum rate reached at a substrate concentration close to the critical micellar concentration, 0.1 mM. 3. 3. In the presence of sodium dodecylsulphate, cholate, deoxycholate and Triton X-100 the hyperbolic kinetics maintained with the maximum rate still reached at 0.1 mM GD1a. These detergents behave as non-competitive inhibitors although Triton X-100, at low concentrations, exhibits an activating effect. 4. 4. Triton QS-31 and Lubrol WX cause a specific change of the enzyme kinetics. With Triton QS-31 the v /[ S ] relationship shifts to a double-shouldered curve with a transition around 0.07–0.08 mM GD1a. With Lubrol WX the v /[ S ] curve is sigmoidal with transition around 0.3 mM and a maximum rate at 1.0 mM GD1a. In the case of Triton QS-31 the addition of albumin restores the hyperbolic kinetics. 5. 5. Under our experimental conditions, brain membrane-bound neuraminidase appears to act on the monomeric form of the gangliosidic substrate. In the presence of Triton QS-31 the enzyme apparently gains the capacity to work on the micellar form of substrate, while the Lubrol WX it recognizes and affects only the micellar form of substrate.

Glycolyl-neuraminic acid in ox brain gangliosides.

IN this communication we report the presence of glycolyl-neuraminic acid in sialic acids from ox brain gangliosides. Our method of analysis consisted of the following six steps: (i) gangliosides were extracted from ox brain grey matter and purified according to a method which we have previously described1; (ii) gangliosides so obtained were hydrolysed in 0.1 N sulphuric acid at 80° C for 2 h, to split off sialic acid; (iii) sialic acid was purified on ‘Dowex’ 2 × 8 column as acetate according to Svennerholm2, and desalted on ‘Amberlite IR–120’; (iv) the solution was dried in vacuum and the residue was hydrolysed in 1 N sulphuric acid for 5 h at 100° C to obtain glycolic acid3; (v) glycolic acid was purified on ‘Dowex’ 2 × 8 column as acetate and eluted with NaCl 2 M; (vi) the glycolic acid was identified by the Eegriwe reaction4 and by gas-chromatography.

Removal of N-acetylneuraminic acid from particulate sialoglycoproteins by endogenous membrane bound neuraminidase in calf brain

BRAIN membranes are rich in sialoglycoproteins (BRUNNGRABER et al., 1967; GOMBOS et al., 1971). A rational approach to the role of these macromolecules in interneuronal and neuronal-glial recognition requires precise information on their metabolism and on the possible modifications they undergo within the membrane. Release of neuraminic acid is especially important. In previous short-term experiments on different animals we were unable to demonstrate a considerable release of protein bound NANA (PF~ETI et al., 1970; TETTAMANTI et al., 1972~) by the action of brain neuraminidase on endogenous particulate sialoderivatives. These reports suggested that brain membrane bound sialoglycoproteins are resistant to endogenous neuraminidase. However, in 1972 HELILMAN and ROUKEMA provided definite evidence that N-acetyl neuraminic acid could be released from endogenous sialoglycoproteins of calf brain homogenate, indicating that the particulate sialoglycoproteins should also be affected by neurarninidase. We therefore decided to ascertain to what extent brain membrane bound sialoglycoproteins are affected by membrane bound neuraminidase (EC 3.2.1 3).

Intraneuronal distribution of cytosoluble neuraminidase in pig brain.

The cell sap obtained from the brain of different animals displays neuraminidase activity (1,2). This enzyme activity has been recently purified and characterized from pig brain and shown to occur in two multiple forms (3).