4-methylumbelliferyl-glucoside and Gaucher-Disease

Fluorogenic 6-acylamino-4-methylumbelliferyl-beta-D-glucosides were found to be poor substrates for the three known human beta-glucosidases, i.e., lysosomal and non-lysosomal glucocerebrosidases and cytosolic broad-specificity beta-glucosidase. However, homogenates of human tissues and human cell types showed significant enzymatic hydrolysis of 6-ethanoylamino-4-methylumbelliferyl-beta-D-glucoside (EMGlc) due to the activity of a hitherto undescribed beta-glucosidase, called here EMGlc-ase. It was shown that the isozyme is hardly active towards 4-methylumbelliferyl-beta-D-glucoside or glucosylceramide. EMGlc-ase exhibits maximal activity at pH 4.5 and 5.0 in the absence and presence of sodium taurocholate respectively. It is a soluble lysosomal enzyme with a discrete isoelectric point of about 5.0. EMGlc-ase is not inhibited by conduritol B-epoxide, is activated by sodium taurocholate and binds strongly to Concanavalin A. This enzyme is not deficient in relation to Gaucher disease.

Topics: Animals; beta-Glucosidase; Chromatography, Agarose; Chromatography, Gel; Fibroblasts; Fluorescence; Gaucher Disease; Glucosides; Glucosylceramidase; Humans; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Isoelectric Point; Kidney; Liver; Lysosomes; Rats; Spleen; Subcellular Fractions; Substrate Specificity

Gaucher disease is an autosomal recessive sphingolipidosis associated with deficient glucocerebroside beta-glucosidase activity. It is a panethnic metabolic disorder, but the carrier frequency is particularly high among Ashkenazi Jews (estimated between 1:12-1:25). In order to establish a reliable and convenient biochemical assay method for differentiating asymptomatic Gaucher carriers from normal individuals, glucocerebroside beta-glucosidase activity was determined in peripheral blood lymphocytes and cultured skin fibroblasts of 11 Gaucher obligate heterozygotes using the authentic nonlabeled sphingolipid substrate N-palmitoyl dihydroglucocerebroside and the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGP). The level of lymphocyte beta-glucosidase activity on the glucocerebroside substrate was observed to range from 42-65% of that of the control mean, and there was no overlap of enzyme activity between the Gaucher heterozygotes and controls. However, when the artificial fluorogenic substrate 4MUGP was used, the level of beta-glucosidase activity in 2 of the Gaucher obligate heterozygotes was noted to overlap with that of the control individuals. Contrary to findings in the lymphocytes, cultured skin fibroblasts appear to be a reliable enzyme source for Gaucher carrier detection even when the artificial fluorogenic 4MUGP substrate was used, as the level of beta-glucosidase activity in all of the Gaucher obligate heterozygotes tested was intermediate and distinctly separated from that of the control persons. Using the lymphocyte glucocerebroside beta-glucosidase assay and fibroblast 4MUGP beta-glucosidase assay methods, we identified the carrier status in 3 other relatives and ruled it out in 4 others. These data suggest that nonlabeled glucocerebroside is a reliable and highly specific substrate for either lymphocyte or fibroblast beta-glucosidase activity assay in identifying asymptomatic Gaucher carriers. Use of the 4MUGP substrate for differentiating Gaucher heterozygotes from control persons, on the other hand, should be restricted to the fibroblast enzyme assay method, as considerable overlap of enzyme activity was noted in lymphocytes.

Topics: Adult; Cerebrosides; Clinical Enzyme Tests; Female; Fibroblasts; Gaucher Disease; Genetic Carrier Screening; Glucosides; Glucosylceramidase; Glucosylceramides; Glycosides; Humans; Hymecromone; Lymphocytes; Male; Pedigree; Quebec; Umbelliferones

Glucocerebrosidase is present in considerable amounts in human urine. The enzyme is stable in concentrated urine for several days when stored at 0 degrees C. Like tissue glucocerebrosidase, the urinary enzyme is inhibited by conduritol B-epoxide and hydrolyses not only glucocerebroside but also the synthetic substrate 4-methyl-umbelliferyl-beta-D-glucoside. The enzyme is deficient in urine from patients with Gaucher disease (type 1). It is possible to discriminate completely between patients with type 1 Gaucher disease and control subjects by measuring the ratio glucocerebrosidase/beta-hexosaminidase in urine. The value of this ratio (mean +/- SE) with the synthetic substrates 4-methylumbelliferyl-beta-glucoside and p-nitrophenyl-beta-N-acetylglucosaminide, respectively, was 34.2 +/- 3.7 (n = 24) in the controls and 2.1 +/- 0.9 (n = 21) in the patients.

Topics: Adolescent; Adult; beta-N-Acetylhexosaminidases; Child; Female; Gaucher Disease; Glucosidases; Glucosides; Glucosylceramidase; Hexosaminidases; Humans; Hymecromone; Inositol; Male; Middle Aged; Substrate Specificity

'Acid' beta-glucosidase of human spleen, from either normal controls or patients with type 1 (adult) Gaucher disease, was incorporated into phosphatidylcholine liposomes. The non-incorporated (soluble) Gaucher-enzyme had a higher apparent molecular weight than had the corresponding control. Liposomal 'acid' beta-glucosidase prepared from Gaucher-spleen was more thermostable than was the corresponding normal enzyme; it was also stimulated by acidic lipids to a much lesser extent. The results suggest that the genetic mutation in type 1 (adult) Gaucher disease has multiple effects on the glycoprotein form of 'acid' beta-glucosidase.

Topics: beta-Glucosidase; Cell Membrane; Concanavalin A; Gaucher Disease; Glucosidases; Glucosides; Hot Temperature; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Liposomes; Molecular Weight; Phosphatidylcholines; Solubility; Spleen

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.

Topics: Animals; beta-Glucosidase; Chemical Phenomena; Chemistry, Physical; Dose-Response Relationship, Drug; Enzyme Activation; G(M1) Ganglioside; Gangliosides; Gaucher Disease; Glucosidases; Glucosides; Hot Temperature; Humans; Hymecromone; Inositol; Kinetics; Liver; Lysosomes; Male; Micelles; Molecular Weight; Rats; Rats, Inbred Strains; Temperature

Studies were undertaken to characterize the beta-glucosidase activity in freshly homogenized liver from Sprague-Dawley rats. About 95% of the total beta-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 beta-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-beta-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 beta-glucosidase from human liver or spleen. These data indicated that the liver of Sprague-Dawley rats contained primarily the lysosomal acid beta-glucosidase ('glucocerebrosidase') and little, if any, 'nonspecific' beta-glucosidase. This, and the fact that about 60% of the rat hepatic beta-glucosidase could be solubilized by autolysis, freezing and rethawing or extraction with water, contrasts with the beta-glucosidases in human liver since about 80% of the total beta-glucosidase activity is cytosolic and does not hydrolyze glucosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; beta-Glucosidase; Gaucher Disease; Glucosidases; Glucosides; Humans; Hymecromone; Liver; Lysosomes; Male; Rats; Rats, Inbred Strains; Solubility; Species Specificity; Subcellular Fractions

This report describes a reliable and reproducible method for the identification of carriers of Type I Gaucher disease using blood platelets as the source of beta-glucosidase and 4-methylumbelliferyl-beta-D-glucoside as substrate. Platelet lysates have at least two identifiable beta-glucosidase activities with the synthetic substrate. One is maximally active at pH 5.0 in the absence of sodium taurocholate and the other at pH 5.6 in the presence of taurocholate. In platelets of Gaucher homozygotes and heterozygotes, the beta-glucosidase activity at pH 5.6 with the bile salt is reduced whereas the activity at pH 5.0 is the same in non-carriers, carriers and affected patients. In addition to differences in specific activity, the ratio of beta-hexosaminidase to beta-glucosidase activities is a useful parameter in the evaluation of the carrier state. Since carriers have normal activity of hexosaminidase and a reduced activity of beta-glucosidase, their mean activity ratio is about 70% higher than in non-carriers. Therefore we propose that the specific activity of beta-glucosidase at pH 5.6 in the presence of sodium taurocholate with the ratio of beta-hexosaminidase to beta-glucosidase serve as useful and reliable indices in the evaluation of the carrier state for Gaucher disease.

Topics: Adult; beta-Glucosidase; Blood Platelets; Gaucher Disease; Genetic Carrier Screening; Glucosides; Hexosaminidases; Homozygote; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Leukocytes; Taurocholic Acid

The description in 1965 of glucocerebroside: beta-glucosidase as the enzymic defect in Gaucher's disease stimulated considerable research interest and effort toward establishing rapid, reliable, and inexpensive enzymic assays for diagnostic purposes and carrier detection. Here, we consider some of the methods currently in use in which the substrate is the synthetic glucoside, 4-methylumbelliferyl-beta-D-glucopyranoside, and leukocytes and fibroblasts are the sources of enzyme. We also consider the concepts of the "acid beta-glucosidase" and multiple forms of beta-glucosidase that have been proposed to explain the effectiveness of the fluorometric assays. Finally, we analyze the limitations of each method and discuss the difficulties involved in instituting heterozygote screening programs in the general population.

Topics: beta-Glucosidase; Clinical Enzyme Tests; Gaucher Disease; Glucosidases; Glucosides; Heterozygote; Humans; Hymecromone; Isoenzymes; Leukocytes

A procedure has been developed for the determination of glucocerebrosidase activity using the substrate analogue, 2-N-hexadecanoylamino-4-nitrophenyl-beta-D-glucopyranoside (HNGlu) with sodium taurocholate and oleic acid as activators. Cultured skin fibroblasts and amniotic fluid cells have been used as the enzyme source. It has been used successfully to confirm the diagnosis of two Type I and two Type II Gaucher patients. The procedure shows approximately a 15-fold increase in sensitivity over other procedures using HNGlu as substrate. Compared with 4-methylumbelliferyl-beta-D-glucoside, HNGlu proves to be a highly specific substrate for glucocerebrosidase with little or no hydrolysis by the beta-glucosidases present in fibroblast extracts. It is therefore the chromogenic substrate of choice for determining a glucocerebrosidase deficiency.

Topics: Cells, Cultured; Fibroblasts; Gaucher Disease; Glucosidases; Glucosides; Glucosylceramidase; Glycosides; Humans; Hymecromone; Methods; Skin; Substrate Specificity

Human leukocytes contain at least two isozymes of 4-methylumbelliferyl-beta-glucosidase acting optimally at pH 4.0 and 4.8; in Gaucher disease, only the former is deficient. Brief exposure of the leukocyte homogenate to pH 4.0 at room temperature results in irreversible inactivation of the pH 4.8 activity, while the activity at pH 4.0 remains unaffected. The more acidic isozyme is stimulated four- to fivefold by 0.2% sodium taurodeoxycholate (TDC) with a shift in the pH optimum to 5.0. The less acidic isozyme is completely suppressed in the presence of this detergent. Both leukocyte isozymes appear to be membrane-bound since gel filtration of Sephadex G-200 produces only one peak of activity located at the void volume, unlike in liver and kidney where a second peak also can be demonstrated. Heat inactivation analysis indicated that in controls, assayed in the absence of detergent, pH 4.0 activity is more thermostable than pH 4.8 activity. However, in Gaucher disease, the residual beta-glucosidase at pH 4.0 is just as thermolabile as the unaffected pH 4.8 activity. Heat inactivation of the enzyme in the presence of TDC resulted in rapid loss of activity, suggesting a direct effect of the bile salt on the configuration of the enzyme decreasing its thermal stability. In the absence of detergent, acid beta-glucosidase shows two K(m)'s, one at 3.2 mM and another at 0.9 mM. In the presence of detergent, only the higher K(m) at 3.3 mM is obtained. In patients with Gaucher disease and in obligate carriers, the K(m) remains essentially unaffected while the V(max) shows the expected deficiency.A reliable and reproducible selective assay technique has been developed for the diagnosis of Gaucher disease homozygotes and obligate heterozygotes and for the carrier screening of individuals at risk for this inherited disorder. The efficacy of this technique has been demonstrated by studying the activity in 42 controls, 26 patients, 32 obligate heterozygotes, and 23 healthy relatives of patients with Gaucher disease.

Topics: Adult; beta-Glucosidase; Child; Chromatography, Gel; Detergents; Female; Gaucher Disease; Genetic Carrier Screening; Glucosidases; Glucosides; Heterozygote; Homozygote; Humans; Hydrogen-Ion Concentration; Hymecromone; Isoenzymes; Kidney; Kinetics; Leukocytes; Liver; Male; Temperature

Leukocytes were isolated from 14 patients (7 males and 7 females ) with Gaucher disease of the Norrbottnian type (Type 3), 32 obligate heterozygotes (16 males and 16 females) for this disease and 20 controls (10 males and 10 females). After collection, the cells were transported in dry ice to the laboratory, where they were assayed. The assays were repeated after the cells had been stored for 12 months. beta-Glucosidase activity was assayed with D-[glucose-U-14C]glucosylceramide at pH 5.8 with Cutscum-Na-cholate as a detergent and 4-methylumbelliferyl-beta-glucoside at pH 4.1 with Triton-Na-taurocholate as a detergent. The activities of two marker enzymes, 4-methylumbelliferyl-beta-galactosidase and N-acetyl-beta-glucosaminidase, were assayed in aliquots of the same leukocyte samples. The activity of beta-galactosidase remained constant during storage, N-acetyl-beta-glucosaminidase increased, while beta-glucosidase decreased as assayed with the natural as well as with the artificial substrate. beta-Glucosidase activity was significantly lower in the female than in male controls and heterozygotes. When assayed with natural substrate beta-glucosidase activity in leukocytes from the male patients was 6--12% of the control mean value and 10--15% in those from the female patients. The corresponding figures found when the artificial substrate was used were 15--30% and 22--45%. The values for the heterozygotes were respectively 42--68% and 34--79% with the natural substrate, and 33--82% and 51--109% with the artificial substrate. No correlation was found between the age of the patient and the beta-glucosidase activity.

Topics: Acetylglucosamine; Acetylglucosaminidase; Adolescent; Adult; Age Factors; Aged; beta-Galactosidase; beta-Glucosidase; Child; Child, Preschool; Drug Stability; Female; Galactosides; Gaucher Disease; Glucosamine; Glucosidases; Glucosides; Glucosylceramidase; Heterozygote; Homozygote; Humans; Hymecromone; Leukocytes; Male; Middle Aged; Sex Factors

1. Cases of Gaucher's disease could not be distinguished from controls by the assay of beta-glucosidase activity in water homogenates of liver using 4-methylumbelliferyl-beta-D glucopyranoside. 2. Two peaks of beta-glucosidase activity were separated by Sephadex G-150 gel filtration in control and Gaucher livers. In the presence of the elution buffer pH profiles of peak I showed a deficiency at pH 3.5-4.5 in Gaucher's disease. Gaucher and control peak II had similar pH profiles with little or no activity at pH 3.0-4.0. 3. A clear distinction between homogenates of Gaucher and control liver was obtained by assay at pH 4.0 in the presence of elution buffer, or of sodium chloride, a component of the elution buffer.

Topics: Gaucher Disease; Glucosidases; Glucosides; Glucuronidase; Humans; Hydrogen-Ion Concentration; Hymecromone; Liver; Umbelliferones