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A biochemical study of three patients with clinical symptoms of Gaucher disease was carried out. Two of them had a significant deficiency of beta-glucocerebrosidase activity (a primary enzyme defect) in leukocytes and an enormous increasing of chitotriosidase activity in blood plasma that confirmed the diagnosis of Gaucher disease. Some differences in stability of mutant enzymes were found in these two cases. Mutation analysis revealed two point mutations--N370S and L444P in beta-glucocerebrosidase gene of both patients. Correlation between clinical picture, peculiarities of enzymatic defect and genetic status of patients is discussed. The influence of some epigenetic factors on phenotypic manifestation of the disease is supposed.

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The mutation analysis of alpha-galactosidase A gene was carried out in two families with Fabry disease described by us earlier. In the family P. a new point mutation E341K (a G to A transition at position 10,999 of the gene) was identified. The mutation causes a Glu341Lys substitution in alpha-galactosidase A molecule. Another point mutation was identified in a patient from family N. who had unusual unusually high residual activity of alpha-galactosidase A. The mutation was identified as R112C (a C to T transition at position 5233 of alpha-galactosidase A gene) and it caused the Arg112Cys substitution in the enzyme molecule. This mutation was earlier described in Japanese patient with showed a complete loss of enzyme activity. However, in this case the mutation was combined with another mutation Glu66Gln. The relationship between genetic heterogeneity and clinical manifestation of Fabry disease is discussed.

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Fourteen members of family P. and four members of family N. were clinico-biochemically examined. Among twelve adult children (19-32 years old) of family P. five sons manifested angiokeratotic skin lesions and other clinical signs of Fabry disease. Three of the probands had additional symptoms not generally found in Fabry disease. Biochemical studies including an enzyme assay, analysis of storage products and alpha-galactosidase multiple forms, allowed us to confirm the diagnosis of Fabry disease in four affected brothers and to establish the heterozygous status of their mother. The data of biochemical investigation of patient N. with atypical variant of Fabry disease are also presented. The patient N. with strong skin lesions had a high residual alpha-galactosidase activity and unusual composition of alpha-galactosidase multiple forms.

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In vitro it was studied the isoform spectra of the intracellular and secreted alpha-L-fucosidase from skin fibroblasts of patients with Fabry disease (glycolipidosis), Hurler and Sanfilippo D diseases (mucopolysaccharodosis, types I and III) and in the normal state was studied. It was shown that the multiple form profile of secreted alpha-L-fucosidase in patients fibroblasts was changed as compared to that in control: the pathological cells were characterized by expression of more basic isoforms of alpha-L-fucosidase. The changes were similar to those in sucrose-loaded normal cells, modelling storage disease. The data obtained allow the suggestion that the intracellular accumulation of compounds whose hydrolysis was disturbed on a hereditary deficiency of enumerated glycosidases can influence the posttranslational processing of alpha-L-fucosidase, the enzyme which is not primary affected in these disorders. These data allow the conclusion that the high phenotypic heterogenity of lysosomic storage diseases is possibly due to the influence of so-called epigenetic factors involving the changes in properties of such glycosidases as are not associated with a primary hereditary defect.

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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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Two cases of Gaucher's disease are described. The diagnosis was based on clinical appearance, observation of the typical cells in the sternal and splenic puncture biopsies. Leucocytic beta-glucocerebrosidase activity was found inhibited in contrast to normal activity of the other lysosomal glycosidases. A female of 51 developed significant changes in the bones, progressive enlargement of the liver, chronic calculous cholecystitis 36 years after splenectomy. Current aspects of pathogenesis, clinical symptoms, diagnosis and treatment of Gaucher's disease are reviewed.

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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 method of hybridization was used to obtain homokaryons of normal fibroblasts, of fibroblasts from children with Tay-Sach's and Sandhoff's diseases (TSD and SD) and heterokaryons of fibroblasts from children with the same diseases. Hexosaminidase A was detected in heterokaryons of fibroblasts from children with TSD and SD by isoelectric focusing. The data pertaining to the heterogeneity of different forms of hexoaminidase are discussed as are the reconstructions of the isozymes depending on the time and index of hybridization. It is stressed that the method of genetic complementation may be used for confirming the diagnoses of TSD and SD and further study of their heterogeneity.

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Activities and isozyme spectra of alpha-L-fucosidase and beta-D-hexosaminidase were similar both in biopsy material from fetal zone of placenta (chorion) and in chorion tissue obtained after abortion. Evaluation of the isozyme spectrum of these glycosidases in biopsy reeterial of chorion might be carried out for prenatal detection of fucosidosis (deficiency of alpha-L-fucosidase), Tay-Sachs disease (deficiency of hexosaminidase A) and of Sandhoff disease (deficiency of hexosaminidases A and B).

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During affinity chromatography on N-(epsilon-aminocaproyl)-beta-L-fucopyranosylaminosepharose of the enzyme preparation of alpha-L-fucosidase from human kidney the elution profile of the enzyme revealed two components, which can be designated as alpha-L-fucosidases A and B. In the absence of sodium aside in the buffer mixtures used one of the components (fucosidase A) was retained by an affinity adsorbent, while the other one (fucosidase B) was adsorbed under the same conditions; the latter component was eluted with a solution containing the enzyme inhibitor--L-fucose. Data from the enzyme rechromatography suggest an equilibrium of the fucosidases A and B, which differ in their affinities for the affinity sorbent.

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The effect of a partially purified preparation of pig kidney alpha-L-fucosidase on some glycoproteins--human and rabbit gamma-globulin, glycoprotein from sheep submaxillary gland and ceruloplasmin--was studied. It was shown that the action of the enzyme of the glycoproteins was not accompanied by a release of fucose. A comparative study of the properties of free and concanavalin A-Sepharose 4B-bound alpha-L-fucosidase was done. The experimental data is indicative of difference in the pH-dependenced and thermostability of these two enzyme forms. It was found that bound alpha-L-fucosidase, similar to the free form, did not split off fucose from the native blood group substances. The data of isoelectric fucosing of alpha-L-fucosidase suggests the existence of enzyme polymorphism.

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