Electrophoretic separation of neutral and acid β-glucosidase isozymes in human tissues

Shafit‐Zagardo, Bridget; Devine, Evelyn A.; Desnick, Robert J.

doi:10.1016/0005-2744(80)90235-1

Cited by 10 publications

(4 citation statements)

References 8 publications

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“…However, consistent differences in properties of the enzyme have not been uniformly demonstrated, so that no adequate differentiation ofsubtype or severity within subtype has been provided (15)(16)(17)(18)(19). Recently, multiple forms of normal and Gaucher disease tissue 83-glucocerebrosidase have been described by isoelectric focusing techniques, but this still has not permitted differentiation of Gaucher disease phenotypes (20)(21)(22)(23)(24)(25)(26).…”

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confidence: 99%

Mutations of glucocerebrosidase: discrimination of neurologic and non-neurologic phenotypes of Gaucher disease.

Ginns¹,

Brady²,

Pirruccello³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

104

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Multiple molecular forms of (3-glucocerebrosidase that permit discrimination between neurologic and non-neurologic phenotypes of Gaucher disease have been identified radioimmunologically in fibroblasts and human brain tissue. In normal human fibroblasts these forms have been shown by NaDodSO4/polyacrylamide gel electrophoresis to have apparent Mr of 63,000 (form A1), 61,000 (form A2), and 56,000 (form B). The Mr 63,000 form may be a precursor of the Mr 56,000 form. Nonneurologic Gaucher disease (type 1) fibroblasts and normal brain tissue are characteristic in that they contain only one major immunoreactive protein, the Mr 56,000 form. In contrast, fibroblast extracts and brain tissue from neurologic Gaucher disease phenotypes contain only the higher molecular weight forms A1 and A2. These data and the low residual activity of the enzyme in all the variants of Gaucher disease suggest that the mutations of fi-glucocerebrosidase are allelic and involve the active site. Fibroblast Culture and Preparation. Skin fibroblasts from normals and Gaucher disease patients were obtained from patient biopsies and from the Human Genetic Mutant Cell Repository. All fibroblasts were cultured to confluency in McCoy's 5A medium supplemented with 10% fetal calf serum and antibiotics. Cells were harvested by trypsinization and washed with culture medium and phosphate-buffered saline (P1/NaCl) at pH 7.2. Fibroblasts stci d in a 50% (vol/vol) mixture of Cryoprotective media (M.A. Bioproducts, Walkersville, MD) and McCoy's SA with 40% fetal calf serum were rapidly thawed at 370C and washed with P1/NaCl at pH 7.2.Fibroblast pellets were suspended in 60 mM potassium phosphate (pH 6.6) containing 0.1% Triton X-100 and were then sonicated at 40 W for 5 sec at 40C. The sonicates were centrifuged at 40C at 48,000 x g for 20 min and the supernatants were stored at -20'C. 5607The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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confidence: 99%

Mutations of glucocerebrosidase: discrimination of neurologic and non-neurologic phenotypes of Gaucher disease.

Ginns¹,

Brady²,

Pirruccello³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

104

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“…1 (freshly ho mogenized). Under these same conditions, less than 25% of the activity of the cytosolic, human hepatic ß-glucosidase was bound to concanavalin A [4], Sixth, when the enzyme from the rat liver cytosol was electrophoresed on cellulose-ace tate strips, its migration was slower than the respective cytosolic enzymes of human liver or spleen [4].…”

Section: Resultsmentioning

confidence: 99%

“…Concanavalin A chromatography and gel electro phoresis were performed by the method of ShafitZagardo et al [4] with the following modifications. Tissue homogenates were applied directly to Cellogel without prior ethanol/chloroform extraction and en zymatic activity was also detected by cutting the cellu lose acetate gel into 5-mm strips, eluting the enzyme into 0.1 mol/1 citrate-phosphate buffer, pFI 5.5, and determining the 4MU-Glc activity as described above.…”

Section: Methodsmentioning

confidence: 99%

Lysosomal ß-Glucosidase of Rat Liver

Fabbro¹,

Desnick²,

Gatt³

1984

Enzyme

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Studies were undertaken to characterize the ß-glucosidase activity in freshly homogenized liver from Sprague-Dawley rats. About 95% of the total ß-glucosidase activity was associated with the particulate fraction, whereas only about 3-7% was found in the cytosol. Storage of fresh liver at room temperature for several hours or repeated freezing and thawing of fresh rat liver prior to homogenization, solubilized 20-30% of the total hepatic ß-glucosidase activity. An additional 30% could be solubilized by extracting the particulate sediments with water or Triton X-100. The enzymatic activity in both the particulate and solubilized fractions optimally hydrolyzed 4-methylumbelliferyl-ß-D-glucoside as well as the glycolipid substrate, glucosylceramide, at an acidic pH. The rates of hydrolysis of either substrate by all subcellular fractions were stimulated by addition of sodium taurocholate or phosphatidylserine. The particulate, cytosolic and solubilized enzymes bound to concanavalin A, were inhibited by conduritol B epoxide and migrated more electronegatively on cellulose acetate than the cytosolic acid ß-glucosidase from human liver or spleen. These data indicated that the liver of Sprague-Dawley rats contained primarily the lysosomal acid ß-glucosidase (‘glucocerebrosidase’) and little, if any, ‘nonspecific’ ß-glucosidase. This, and the fact that about 60% of the rat hepatic ß-glucosidase could be solubilized by autolysis, freezing and rethawing or extraction with water, contrasts with the ß-glucosidases in human liver since about 80% of the total ß-glucosidase activity is cytosolic and does not hydrolyze glucosylceramide. Thus, rat liver ß-glucosidase provides a useful source for studies of the lysosomal ß-glucosidase, uncontaminated with other enzymes of similar activity. Also, the fact that a major portion of the enzyme could be solubilized without the use of detergents suggests that in rat liver the lysosomal ß-glucosidase is not an integral constituent of the lysosomal membrane and provides a source of soluble enzyme free of detergent.

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“…Samples of ~-D-mannosidase were prepared from the leukocytes and liver by the method of Schafit-Zagardo et al (1980) and volumes of 3 ,~1 of each sample were subjected to electrophoresis on a cellulose acetate membrane (Fluharty et aI., 1971). The isozyme pattern was examined by measurement of the fluorescence at pH 3.5, pH 4.5 and pH 6.5, as described by Poenau and Dreyfus (1973).…”

Section: Methodsmentioning

confidence: 99%