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The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Gal beta 1-4GlcNAc beta 1-R fragment were subjected to enzymic fucosylation. The isomer with a 1-3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated whereas Fuc alpha 1-2Gal beta 1-4GlcNAc displayed the same substrate properties as Gal beta 1-4GlcNAc. FT from cerebellum and forebrain was shown to have the specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from forebrain, the specificity profiles being similar.

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alpha-L-Fucosidase (EC 3.2.1.51) has been isolated from human kidney and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose and fucosylamino-Sepharose. The catalytic activity and oligomeric structure of the enzyme were studied in a reversed micelle system of aerosol OT in octane. Depending on the degree of hydration (a parameter determining the geometrical sizes of the inner aqueous cavity of micelles), fucosidase is present within micelles as 53 kDa monomers, 110 kDa dimers, 230 kDa tetramers and 480 kDa octamers. Association of the monomers into tetra- or octamers causes a 3-4 fold increase in the specific catalytic activity of alpha-L-fucosidase. At pH and ionic strength values corresponding to intralysosomal ones alpha-L-fucosidase is isolated from tissues exclusively in a tetrameric form. After treatment with sodium cholate and subsequent dialysis this tetramer irreversibly dissociates into monomers; this reaction is accompanied by 2-3-fold decreases in the specific catalytic activity of alpha-L-fucosidase. The enzyme tetrameric structure and specific catalytic activity may be reconstituted in a reversed micelle system in the presence of glycolipids-di- and trihexosylceramides, GM1-ganglioside and a mixture of bovine brain gangliosides.

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Kinetic of hydrolysis, by lysosomal glycosidases, of their synthetic substrates were studied in systems of the Aerosol OT (AOT) reversed micelles in octane. Catalytic activity of all the tested enzymes, viz., GM1-galactosidase, beta-hexosaminidases A and B, neuraminidase, and galactocerebrosidase, in reversed micelles proved to be the same as or higher than in the water buffer. In the reversed micelles an effective inhibition of the enzymatic reactions by the resulting carbohydrates was however observed. The dependence of the enzymes' activity on the hydration degree was represented by curves with one or several maxima, corresponding to various oligomeric forms of the enzymes. Dependencies of effective Km on the hydration degree in reversed micelles are similar dependencies of the enzyme activities on the same parameter, which can be explained by corresponding changes of local substrate concentration near the enzyme active site. Dependencies of the enzymatic activity on the surfactant's concentration in the reversed micellar system were also studied. Catalytic activity of the soluble lysosomal glycosidases was found to be unaffected by the micelles concentration. Activity of the membrane lysosomal glycosidase, galactocerebrosidase, strongly increased when the surfactant's concentration decreased. Under optimal conditions the activity of galactocerebrosidase in reversed micelles was 10-fold as compared with its activity in the water buffer.

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The organization of the system of the vimentin intermediate filaments (IFs) in human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis) and their modelling has been studied in vitro. It was shown that during accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal compartment is accompanied by formation of ring-shaped bundles IFs, surrounding apparently these increased organelles. The changed organization of IFs is characteristic of polarised pathological cells in monolayer, and after repassage it is retained only at the spreading state; on transition from the discoid to extended cellular form there occurred the centrifugal shift of ring-shaped structures of IFs to active cell border and gradual restoration of radial fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit compounds reorganization of the vimentin-type IFs in ring-shaped structure is necessary for optimal distribution and stabilization into the cytoplasm of large amounts of increased lysosomes with exo- and endogenous contents. In condition of free spreading (i. e. with diminished cell density) the restoration of normal fibrillar IF organization may be due to the loss of considerable number of hypertrophied lysosomes; the involvement of lysosomal membrane in formation of active cellular border is not to be ruled out.

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Presented are the results of measurements of pH in cytoplasm and lysosomes of skin fibroblasts of healthy donors and patients with lysosomal storage diseases, mannosidosis, Fabry, Krabbe disease. The pH value was estimated in the stationary phase of growth using neutral red (lysosomes) and fluorescein diacetate (cytoplasm). It was shown that the cytoplasmic pH value in pathological cells didn't virtually differ from the control values. The intralysosomal pH value in fibroblasts of patients with mannosidosis and Fabry disease was essentially increased, which correlated with the size increase of these organelles upon the accumulation of unsplit compounds. This led to the decrease in pH gradient between the cytoplasm and lysosomes in the pathological cells, an increase in intralysosomal pH along with hereditary deficiency of enzymes could bring about the retardation of catabolic processes in lysosomes.

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The regulation of the catalytic activity and supramolecular organization of human kidney Gm1-galactosidase and neuraminidase was investigated in the reversed micellar systems of Aerosol OT in octane. It was found that in the reversed micellar systems the Gm1-galactosidase can exist in the monomeric, tetrameric or octameric forms depending on the H2O/surfactant ratio in the system which determines the micelle size. The association of Gm1-galactosidase monomers into octameric structure characteristic of Gm1-galactosidase in the lysosomes results in a two fold increase of the specific catalytic activity of the enzyme. 32 kDa "protective" protein--the component of Gm1-galactosidase--neuraminidase native complex was found to improve significantly this association.

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The changes in intralysosomal pH were measured in the stationary phase of normal human embryonic fibroblast growth under sucrose loading over a period of 6 to 120 hours and in cells with a typical lysosomal storage pathology, Fabry's disease, using a vital indicator dye, neutral red. It was shown that long-term hypertrophy of the lysosomal compartment during intracellular accumulation of non-hydrolysable compounds is concomitant with a pH increase, on the average, by 0.4 units. The highest values of pH (7.0-7.2) were seen in large-sized heterogeneic lysosomes of pathological cells. It is suggested that an increase in intralysosomal pH during accumulation of non-hydrolysable compounds leads to deterioration of conditions that are favourable for the acidic hydrolase function.

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It was shown that human alpha-D-galactosidase is represented by multiple forms, only one of which can also split alpha-D-fucoside. Fabry's disease was found to be associated not only with the deficiency of the alpha-D-galactosidase total activity but also with the deficiency of the alpha-D-fucosidase activity. The decrease in the alpha-D-galactosidase activity is due to the lack of two enzyme forms, while the profile of alpha-D-fucosidase multiple forms during isoelectric focusing of human enzyme preparations is modified very little in comparison with the normal one. The deficiency of both enzymes was expressed in most degree in leukocytes as compared to other tissues. The residual activities of alpha-D-galactosidase and alpha-D-fucosidase in leukocytes were equal to 3.5 and 21%, respectively. Since the decrease in the alpha-D-fucosidase activity was not so noticeable as in the alpha-D-galactosidase activity, it may be expected that the determination of the alpha-D-fucosidase activity can no longer be regarded as a reliable test for the diagnosis of Fabry's disease. The data obtained suggest that alpha-D-galactoside and alpha-D-fucoside are split by the same enzyme, the multiple forms of which are characterized by selective specificity towards these substrates.

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Intracellular activation of lysosomal glycosidases from human skin fibroblasts (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) was shown to occur on the 3rd-6th days of cultivation in media containing 0.04 M sucrose. The increase in the enzyme activity ranged from 40 to 300% depending on cell strain, nature of enzyme and cultivation time. Among pre- and postnatal fibroblast strains, those with a high and low response to sucrose load were identified. The maximal intracellular activation was observed in beta-D-galactosidase, the minimal one--in beta-D-glucuronidase. In pathological cells (Krabbe's disease) the highest activation by sucrose load was observed, as in normal cells, with beta-D-galactosidase, whereas the lowest one--with beta-D-glucuronidase. Secretion of lysosomal glycosidase is selective and noncoordinated. The maximal secretion of alpha-L-fucosidase and beta-D-hexosaminidase was observed within the first 24 hours (intensive sucrose endocytosis), but was considerably decreased at later times, i. e., by the 3rd and 6th days. The enzymes secreted during the 1st and 3rd days differed significantly in stability (37 degrees C, pH 7.0).

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Three fractions of glycolipids--monohexosylceramide, dihexosylceramide (DHC) and trihexosylceramide (THC) were isolated from kidney of patient with Fabry disease. As compared with normal state amount of DHC and THC was increased in the patient kidney 9-19-fold and 15-26-fold, respectively. Gas liquid chromatography showed that the DHC fraction consisted in digalactosylceramide, while the THC fraction--a mixture of digalactosylglucosylceramide (90%) and trigalactosylceramide (10%). Presence of the latter glycolipid was not early found in human body both in normal state and in Fabry disease. Accumulation of DHC and THC was also detected in urine precipitates of the patients using thin-layer chromatography, whereas these substances were not found in urine of one of the patients daughter, who was heterozygote gene carrier of Fabry disease. The data obtained corroborate the Fabry disease presence, which have been predetermined by means of clinical diagnosis as well as basing on deficiency of alpha-D-galactosidase in blood plasma and leukocytes.

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The properties of beta-galactocerebrosidase from human chorionic villi, cultured chorionic villi and cultured skin fibroblasts were compared, using 6-hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGaL) as substrate. The effects of bile salt and Triton X-100 on beta-galactocerebrosidase were examined. It was shown that optimization of the HMGaL assay system requires the presence of pure sodium taurocholate and Triton X-100 at concentrations of 4.5 mM and 0.28 mM, respectively. The optimal pH value was found to be equal to 4.5-5.0; Km for the substrate was 0.03 mM. A comparison of beta-galactocerebrosidase from chorionic villi and cultured chorionic villi with the enzyme from skin fibroblasts revealed the similarity of some properties of these enzymes. The experimental results suggest that HMGaL can be used as a substrate for the identification of chorionic villi beta-galactocerebrosidase in an early prenatal diagnosis of Krabbe's disease.

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On the basis of o-acylamino-4-methylumbelliferon, a number of beta-galactosides and beta-glucosides have been synthesized. The fluorogenic compounds obtained differ by the length of acyl residues. 6- and 8-hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranosides (6-HMGal and 8-HMGal) are shown to be substrates for human galactocerebroside-beta-D-galactosidase. 6-HMGal analogues with shorter acyl residues, octanoyl (OMGal) and butanoyl (BMGal), were cleaved by another type of beta-galactosidase, GM1-ganglioside-beta-galactosidase. It has been established that 6-hexadecanoylamino-4-methylumbelliferyl-beta-D-glucopyranoside (HMGlc) is cleaved by human and animal glucocerebrosidase much slower than its chromogenic analogue (HMGlc). OMGlc did not exceed HNGlc either, though it is cleaved by glucocerebrosidase faster than HMGlc.

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Activity of several lysosomal hydrolases was studied in skin fibroblasts obtained from two brothers living in GDR. Both patients exhibited distinct clinical symptoms of severe neurovisceral disease. Analysis of the lysosomal enzymes activity enabled to exclude possible occurrence in the patients of such glycolipidoses as Gaucher's disease, Sandhoff's disease, GM1-gangliosidosis and metachromatic leukodystrophy. A new fluorogenic galactoside of lipid nature 6-hexadecanoylamine-4-hethylumbellipheryl-beta-D-galactoside used as a substrate of galactocerebrosidase enabled to detect in the patients distinct decrease in this enzymatic activity and to diagnose Krabb's disease. Biochemical diagnosis of Krabb's disease using the fluorogenic substrate was also confirmed by analysis with labelled galactocerebroside as a substrate.

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Main steps are considered of posttranslational modification of lysosomal hydrolases, which are glycoproteins. Processing of the enzymatic carbohydrate moiety in various compartments of endoplasmic reticulum and Golgi apparatus is discussed. Importance of mannose-6-phosphate groups formed during the processing is revealed by studies on binding of these enzymes with specific receptor responsible for their transport into lysosomes. Specificity of lysosomal glycosidases and their isoforms, catalyzing hydrolysis of carbohydrate chains of glycoconjugates and of synthetic substrates dissimilar in the structure, is discussed. Complex structural organization of these enzymes in lysosomes (protein activators and stabilizing factors, presence of marker sites etc) was studied using as an example lysosomal diseases of accumulation, glycosidoses, developed in hereditary deficiency of glycosidases. The data on elevated activity of the majority of lysosomal enzymes in glycosidoses, which are not involved in the primary genetic defect, suggest the possibility of general unspecific response of cells to accumulation of unhydrolyzed compounds.

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Activity of several lysosomal enzymes was studied in leukocytes, blood plasma and skin fibroblasts of two adult brothers with clinical diagnosis of Fabry disease. Activity of ceramide trihexoside-galactosidase was distinctly decreased in both patients. The residual enzymatic activity constituted 5-6% in the patients leukocytes, less than 10% in blood plasma and 25% in fibroblasts as compared with controls. Differences in composition of alpha-D-galactosidase multiple forms were detected in fibroblasts and blood cells of the patients with Fabry disease as compared with normal leukocytes by means of isoelectric focusing.

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The amount of alpha-L-fucosidase secreted by normal human fibroblasts was higher in the medium containing 10% bovine serum than in the medium containing 0.1% bovine serum. Glycosidase secretion was twice higher at the advanced than at the initial stage of subcultivation. Extracellular activity of alpha-L-fucosidase from 3 different fibroblast strains differed insignificantly in the medium containing 0.1% bovine serum, while intracellular activity of the enzyme in these strains was altogether different. The results suggest that the lysosomal glycosidase secretion is determined by the level of cellular endocytosis.

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The intracellular activities of four lysosomal glycosidases (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) in human skin fibroblasts cultured in a medium with 0.1% serum increased in a greater degree than that in a medium with 10% serum. Only two glycosidases (alpha-L-fucosidase and beta-D-hexosaminidase) were secreted by fibroblasts in the culture medium. The extracellular activity of alpha-L-fucosidase and beta-D-hexosaminidase was equivalent to 80 and 25% of their intracellular activity in serum-sufficient fibroblasts and 40 and 15%--in serum-restricted fibroblasts. These results suggest that the observer phenomena are controlled by the levels of autophagy, endocytosis and membrane recycling.

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