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We have studied the substrate specificities of a non-specific activator protein on the enzymatic hydrolyses of the following compounds: GM1 and GM2, as well as several of their derivatives including oligosaccharides, GgOse3Cer-II3-sulfate and LacCer-II3-sulfate, Gb-Ose3Cer and GbOse4Cer, three neolacto-series glycosphingolipids, and two non-ceramide glycolipids. Our results show that this activator protein has a broad spectrum of activity and exhibits the properties of a nonspecific natural detergent. The evidence of non-specificity was the ability of this activator protein to stimulate the hydrolyses of glycolipids, regardless of glycosphingolipids or non-ceramide glycolipids, carried out by glycosidases from animals, plants, and microorganisms. Its activity was, however, limited to substrates that had a lipid moiety. The oligosaccharide of GM1 and deacetyl-fatty acid free GM1 (II3-NeuGg-Ose4-sphingosine) were hydrolyzed by beta-galactosidase in the absence of this activator protein.

beta-D-N-Acetylglucosaminidase isozymes were separated and assayed in the plasma of control healthy individuals and pregnant women by an automated method consisting in chromatofocusing on polybuffer exchanger PBE-94 column, flow-through fluorimetric determination of activity and computer assisted quantification. Under the established optimal conditions the method fractionated beta-D-N-acetylglucosaminidase into four isozymes. A, I2, I1 and B, with the analytical coefficients of variation of 1.8, 2.2, 6.4 and 4.1%, respectively. Duration of a single analysis was 25 min including washing, and 10-15 successive runs could be performed on the same column with good reproducibility. A linear activity response was observed from 1-5 microliters of plasma (depending on the individual isozyme) to 50 microliters, and the detection limit was 0.016 mUnits. Isozyme A was heat labile. Upon sialidase treatment, isozymes A, I2 and I1 released sialic acid and were eluted from the column at less acidic pHs. In healthy individuals isozymes A, I2, I1 and B covered about 62.8, 6.9, 15.0 and 15.1% of the total beta-D-N-acetyl-glucosaminidase activity, respectively. During pregnancy the plasma concentration of all isozymes increased. Isozyme I2 showed the highest enhancement (30-fold), followed by I1 (8-fold), B (5.6-fold) and A (3-fold). Interruption of pregnancy by either physiological delivery or ambulatory abortion was followed by a sharp fall of the concentration of all isozymes reaching, in a few days, the control levels.

A number of data of kinetics nature indicate that brain neuraminidases (the cytosol and the membrane bound enzyme) recognize the physical state of gangliosidic substrate, with immediate modification of the activity. The interactions between the enzyme(s) and the different physical forms of the substrate are still to be studied at the molecular level. The knowledge of these phenomena would greatly help understanding the role played by gangliosides in the neuronal plasma membrane.

A female patient of Italian, non-Jewish, descent, 22 years of age, with cloudy corneae, capsular lens opacities and severe and progressive mental and motor deterioration is described. Ultrastructural examination of a skin biopsy sample showed storage of membranous cytoplasmic bodies in Schwann cells, vessel walls, fibroblasts, smooth muscle fibres and sweat glands, and the presence of some electron-lucent vacuoles, filled with fibrillo-granular material, in sweat glands. Biochemical analysis of cultured fibroblasts (from skin explant) showed a complete deficiency of the sialidase acting on gangliosides, while the sialidase acting on sialyllactose and MU-NeuAc, and several lysomal hydrolases were normal. The urine sediment analysis showed accumulation of all phospholipid species, of several glycolipids and of gangliosides, especially of the polysialylated species. We conclude that the patient under examination is affected by Mucolipidosis IV and the term 'sialolipidosis' is suggested for this inborn disorder.

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.

Publisher Summary This chapter discusses a study to examine changes in rabbit brain cytosolic and membrane bound gangliosides during prenatal life. The developmental profile of cytosolic and membrane bound gangliosides in rabbit full brain from the 21st day of pregnancy till birth was studied. The patterns of the individual membrane bound and cytosolic gangliosides, in the same period of life, were very similar. At 21 days of pregnancy, the most abundant gangliosides were GTlb and GQlb, followed by GDla and GDlb; at birth, it was GDla followed by GTlb, GDlb, and GM1. The ganglioside pattern in the early rabbit fetus brain, characterized by the abundance of multisialogangliosides, closely resembling that of human fetus, is somewhat similar to that occurring in adult brain in some fish and amphibian species. This suggests possible links, with regards to gangliosides, between brain phylogenesis and mammalian brain ontogenesis.

The binding of ganglioside GM1 to bovine serum albumin has been studied by using absorption and fluorescence properties of the protein chromophores. Differences in the ultraviolet absorption spectrum and in fluorescence quenching, as well as a marked shift of the wavelength at the fluorescence maximum provide information about the binding of this ganglioside to albumin. Ultracentrifugal studies showed that there are two forms of the GM1-protein complexes which differ markedly in their molecular weight. These two forms have been separated on this basis, by a chromatographic sieving procedure, and designated as complexes I and II. Both complexes are characterized by a GM1: protein ratio of one ganglioside micelle per albumin polypeptide chain. Complex II polymerizes slowly and irreversibly to a dimer, complex I. These results have been correlated with the optical studies in order to draw limited inferences as to the environment of the binding sites on the native protein. The interaction between GM1 micelles and albumin is mostly hydrophobic and the two complexes are actually mixed ganglioside-protein micelles. At submicellar concentrations of ganglioside a binding of ganglioside GM1 to albumin also occurs. This process is due, however, to an aspecific, reversible adhesion of GM1 molecules on the albumin surface with no apparent perturbation of the albumin structure.

We have studied the substrate specificities of a non-specific activator protein on the enzymatic hydrolyses of the following compounds: GM1 and GM2, as well as several of their derivatives including oligosaccharides, GgOse3Cer-II3-sulfate and LacCer-II3-sulfate, Gb-Ose3Cer and GbOse4Cer, three neolacto-series glycosphingolipids, and two non-ceramide glycolipids. Our results show that this activator protein has a broad spectrum of activity and exhibits the properties of a nonspecific natural detergent. The evidence of non-specificity was the ability of this activator protein to stimulate the hydrolyses of glycolipids, regardless of glycosphingolipids or non-ceramide glycolipids, carried out by glycosidases from animals, plants, and microorganisms. Its activity was, however, limited to substrates that had a lipid moiety. The oligosaccharide of GM1 and deacetyl-fatty acid free GM1 (II3-NeuGg-Ose4-sphingosine) were hydrolyzed by beta-galactosidase in the absence of this activator protein.

The possible occurrence of circadian and circannual rhythms in the plasma concentrations of the following enzymes of lysosomal origin was assessed: beta-D-N-acetylglucosaminidase (EC 3.2.1.30) beta-D-glucuronidase (EC 3.2.1.31), beta-D-glucosidase (EC 3.2.1.21), beta-D-galactosidase (EC 3.2.1.22), alpha-D-galactosidase (EC 3.2.1.23), alpha-L-fucosidase (EC 3.2.1) and alpha-D-mannosidase (EC 3.2.1.24). The circadian rhythm was studied in 16 women (aged: 17-24 years) and 13 men (age: 23 years) volunteers; the circannual rhythm, in 10 women and 8 men (age: 20-25 years). The circadian rhythm was detected in all the tested enzymes of women, and only in alpha-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase and beta-D-acetylglucosaminidase of men. A statistically significant difference between genders in the circadian rhythm was exhibited by beta-D-galactosidase (MESOR; amplitude) beta-D-glucosidase (MESOR; amplitude; acrophase) beta-D-N-acetylglucosaminidase, beta-D-glucuronidase and alpha-D-galactosidase (MESOR) and alpha-L-fucosidase (amplitude, acrophase). A circannual rhythm was detected in all the tested enzymes with the exception of beta-D-glucuronidase and beta-D-N-acetylglucosaminidase; no statistically significant difference between genders was detected. The group rhythms of some of the enzymes (alpha-D-galactosidase, beta-D-glucosidase, beta-D-galactosidase) showed similar values of both circadian and circannual acrophases, suggesting that they may subjected as a group to the same chronobiological coordination, possibly mediated by hormones. The chronobiological rhythms of lysosomal enzymes were different from those of lactate dehydrogenase and alpha 1-antitrypsin, indicating that these rhythms are not merely reflecting fluctuations of the water content of plasma. No in-phase relationship was observed between the circadian and circannual rhythms of plasma cortisol and those of the tested lysosomal enzymes, excluding a direct chronobiological and possibly functional relationship between this hormone and lysosomal enzymes.

New methods have been developed for the preparation of highly purified gangliosides, homogeneous in the saccharide, long chain base, and fatty acid moieties and gangliosides carrying different kinds of labelled probes. Gangliosides, homogeneous in the oligosaccharide portion, were prepared by preparative normal phase HPLC on a Lichrosorb-NH-2 column, using a gradient of acetonitrile-phosphate buffer, pH 5.6, as solvent system. Each class of ganglioside (from monosialo- to tetrasialogangliosides) was then submitted to reversed phase HPLC on a preparative RP-8 column, using acetonitrile-5 mM phosphate buffer, pH 7, as solvent system, to obtain gangliosides homogeneous in the long chain base moiety. Gangliosides containing C18 and C20 sphinganine were prepared by catalytic hydrogenation of the corresponding unsaturated gangliosides. GM1 with homogeneous acyl chain was prepared by alkaline hydrolysis in the presence of tetramethylammonium hydroxide (which forms a GM1 deacetylated at the level of sialic acid, and a GM1 deacetylated at the level of sialic acid and deacylated at the level ceramide), followed by re-N-acylation, carried out in the presence of dimethylaminopropyl, ethylcarbodiimide and natural fatty acids, or of mixed anhydride of ethylchloroformate and 14C-stearic acid, and re-N-acetylation performed with acetic anhydride or labelled acetic anhydride. The GM1 derivative, de-acetylated at the level of sialic acid, also produced by alkaline treatment of GM1, was submitted to re-N-acetylation with 14C-acetic anhydride to produce specifically 14C-labelled GM1. Re-N-acylation was carried out a) in the presence of dimethylaminopropyl, ethylcarbodiimide and natural fatty acids, b) with mixed anhydride of ethylchloroformate and 14C-stearic acid. After re-N-acylations, re-N-acetylation was performed with acetic anhydride or labelled acetic anhydride. Gangliosides tritium labelled in the oligosaccharide moiety were prepared by the galactose oxidase/3H NaBH4 method, and gangliosides tritium labelled at carbon-3 of unsaturated long chain bases by the dicyano-dichlorobenzoquinone (DDQ)/3H NaBH4 method.

The specific activities of five glycohydrolases of lysosomal origin (beta-N-acetylglucosaminidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase and alpha-fucosidase) were measured in different types of primary and metastatic tumors of the human nervous system. The activities of these hydrolytic enzymes in samples of tumor tissue were compared with those in the white matter of 'control' tissue. The specific activities of beta-glucuronidase and beta-N-acetylglucosaminidase were significantly higher (P less than 0.001) in each group of tumors than in normal cerebral matter. The activities of the other hydrolases were sometimes significantly increased in primary tumors, but not always. In metastatic tumors, they were also significantly higher (P less than 0.01).

In the adult Quaking mutant, there is an increase in some polysialogangliosides (GT1 and Gq) and a 50% decrease in BM1 content. Thus, the latter ganglioside, which is a constituent of mature myelin, could be linked to one of the late steps of the myelination process. A novel ganglioside ALG1, which appears during the postnatal development of brain, has been isolated and purified; it contains an alkali labile linkage and after alkaline treatment yields GT1b. Comparison between normal Mice and Quaking mutants does not show any significant quantitative differences, under our conditions. At the stage at which it appears, it could be linked to steps of brain maturation other than myelimation.

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.

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.

Gangliosides are glycosphingolipids containing sialic acid (primarily N-acetyl-neuraminic acid). They are present in brain in different forms, which differ in the pattern of fatty acids and sphingosines, and in the number and sequence of units forming the carbohydrate portion of the molecule. The main series of known gangliosides are distinguished on the basis of the common neutral glycosphingolipid moiety (*): a) G 3 series, galac-tosyl-β(1–4)glucosyl-β(1–1)ceramide; b) G 2 series, N-acetyl-galactosaminyl-β(1–4)galactosyl-β(1–4)glucosyl-β(1–1)ceramide; c) G 1 series, galactosyl-β(1–3)N-acetyl-galactosaminyl-β(1–4)galactosyl-β(1–4)glucosyl-β(1–1)c e-ramide. The G 3 and G 2 series contain gangliosides having one (GM3 and GM2) or two (GD3 and GD2) residues of sialic acid; the G 1 series contains gangliosides having one (GM1), two (GD1a and GD1b), three (GT1a and GT1b), and four (GQ1) residues of sialic acid.

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.