A revised fluorometric assay for Gaucher's disease using conduritol-β-epoxide with liver as the source of β-glucosidase

Daniels, L B; Glew, Robert H.; Radin, Norman S.; Vunnam, Ranga R.

doi:10.1016/0009-8981(80)90168-0

Cited by 39 publications

(18 citation statements)

References 17 publications

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“…TC or PS greatly increased the effectiveness of CBE inhibition of the the enzyme by binding covalently to an amino acid at the catalytic site [13,14], these results suggested that TC or PS facilitated the interaction of the hydrophilic CBE with the catalytic site [16]. Figure 4 compares the CBE inhibition of the ß-Glc from normal and Gaucher disease type-1 (Ashkenazi-Jewish) lymphocyte ex tracts.…”

Section: Effects Of Tc or Psmentioning

confidence: 99%

“…Several compounds which inhibit ly sosomal ß-glucosidase activity also have been identified. These include: conduritol B epox ide (CBE), an inhibitor which binds covalent ly to the catalytic site [13,14], and the two positively charged analogs of glucosyl cera mide; sphingosyl-1-O-ß-.D-glucoside (glucosyl sphingosine [5,15]), and its N-hexyl deriva tive (N-hexyl-glucosyl sphingosine [15]). …”

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confidence: 99%

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Use of Activators and Inhibitors to Define the Properties of the Active Site of Normal and Gaucher Disease Lysosomal ß-Glucosidase

Gátt¹,

Dinur²,

Osiecki³

et al. 1985

Enzyme

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Lysosomal ß-glucosidase (‘glucocerebrosidase’) in peripheral blood lymphocyte and spleen extracts from normal individuals and Ashkenazi-Jewish Gaucher disease type-1 patients were investigated using several modifiers of glucosyl ceramide hydrolysis. The negatively charged lipids, phosphatidylserine and taurocholate, had differential effects on the hydrolytic rates of the normal and Gaucher disease enzymes from either source. With the normal enzyme, either negatively charged lipid (up to 1 mmol/1) increased the reaction rates, while decreasing hydrolytic rates were obtained at greater concentrations. In comparison, the peak activities of the Gaucher enzymes were observed at about 2-3 mmol/1 or 5-8 mmol/1 of phosphatidylserine or taurocholate, respectively. These negatively charged lipids altered only the velocity of the reactions; the apparent K(m) values were not affected. Taurocholate or phosphatidylserine also facilitated the interaction of the normal enzyme with conduritol B epoxide, a covalent inhibitor of the catalytic site. Compared to the normal enzyme, the Ashkenazi- Jewish Gaucher type-1 enzyme required about 5-fold greater concentrations of conduritol B epoxide for 50% inhibition. Neutral or cationic acyl-ß-glucosides were found to be competitive or noncompetitive inhibitors of the enzymes, respectively. Alkyl ß-glucosides were competitive (or linear-mixed type) inhibitors of the normal splenic or lymphocyte enzyme with competitive inhibition constants (K(i)) inversely related to the chain length. With octyl and dodecyl ß-glucoside nearly normal competitive K(i) values were obtained with the splenic enzymes from Gaucher patients. These K(i) values were not influenced by increasing phosphatidylserine or taurocholate concentrations. In contrast, the cationic lipids, sphingosyl-l-Oß- D-glucoside (glucosyl sphingosine) and its N-hexyl derivative, were noncompetitive inhibitors whose apparent K(i) values for the normal enzyme were 30 and 0.25 pmol/1, respectively. The K(i) values for these sphingosyl glucosides were about increased 5 times for the Gaucher type-1 enzymes from Ashkenazi-Jewish Gaucher disease type-1 patients. The K(i) values of glucosyl sphingosine for the normal or mutant enzymes were directly related to increasing concentrations of phosphatidylserine or taurocholate. These data conform to the hypothesis that the active site of human lysosomal ß-glucosidase is composed of at least three domains: (l)a catalytic site which splits the ß-glucosidic linkage; (2) an ‘aglycon binding site’ which binds the ceramide (N-acyl-sphingosyl) residue of the substrate, and (3) a third domain which interacts with the negatively charged lipids (e.g., phosphatidylserine) and the cationic sphingosyl glucosides, thereby altering enzyme catalysis by modulation of V(max). This latter site appears to be specifically altered by a mutation in the structural gene for lysosomal ß-glucosidase in the Ashkenazi-Jewish form of type-1 Gaucher disease.

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Section: Effects Of Tc or Psmentioning

confidence: 99%

mentioning

confidence: 99%

Use of Activators and Inhibitors to Define the Properties of the Active Site of Normal and Gaucher Disease Lysosomal ß-Glucosidase

Gátt¹,

Dinur²,

Osiecki³

et al. 1985

Enzyme

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“…namely, 80% to 85% 30. Despite this profound deficiency of glucocerebrosi¬ dase, this patient had no signs what¬ soever of CNS dysfunction and showed no accumulation of Gaucher cells within the brain.…”

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confidence: 98%

Brain Glucocerebrosidase in Gaucher's Disease

Daniels¹,

Coyle²,

Glew³

et al. 1982

Archives of Neurology

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“…The "-glucocerebrosidase (GCase) activity was determined essentially as described by Wenger and Williams (1991), using the artificial fluorogenic substrate 4-methylumbelliferyl-"-D-glucopyranoside (Sigma M 3633). The GCase activity was measured in the presence and absence of conduritol B-epoxide (CBE) and the difference between these two determinations represents "-glucocerebrosidase activity (Daniels et al 1980).…”

Section: Enzyme Biochemistrymentioning

confidence: 99%

Autopsy case of Gaucher disease type I in a patient on enzyme replacement therapy. Comments on the dynamics of persistent storage process

Hůlková

Ledvinová

Poupětová

et al. 2009

J of Inher Metab Disea

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We report a female patient with Gaucher disease (GD) type I on ERT (imiglucerase) for 5 years, which led to a significant general improvement. Aged 59 years she underwent an episode of altitude sickness followed by sepsis, disseminated intravascular coagulation, and multiorgan failure. She succumbed to a cerebral haemorrhage. Autopsy revealed liver cholestatic cirrhosis and multifocal liver carcinoma with immunophenotype compatible with cholangiocarcinoma. Analysis of the storage process revealed its absence or very low levels in the majority of liver and spleen macrophages. Gaucher cells (GCs) were seen only as occasional aggregates of various sizes in these organs. GCs were seen also in the leptomeninx of the cerebellum and as infrequent perivascular clusters in both the grey and white cerebral matters. Bone marrow was heavily infiltrated with GCs, especially in the adipocyte-rich part. GCs in this location displayed varied degrees of cytoplasmic vacuolation unrelated to the lysosomal compartment, caused by droplets of triglyceride, and interpreted as due to resorption of fragments of altered white adipocytes. All these observations point to the relative efficacy of ERT in covering the standard substrate load, which should not be exceeded as it would lead to the evolution of mature GCs. The results are discussed in relation to our recently published hypothesis on GD cell pathology.

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A revised fluorometric assay for Gaucher's disease using conduritol-β-epoxide with liver as the source of β-glucosidase

Cited by 39 publications

References 17 publications

Use of Activators and Inhibitors to Define the Properties of the Active Site of Normal and Gaucher Disease Lysosomal ß-Glucosidase

Use of Activators and Inhibitors to Define the Properties of the Active Site of Normal and Gaucher Disease Lysosomal ß-Glucosidase

Brain Glucocerebrosidase in Gaucher's Disease

Autopsy case of Gaucher disease type I in a patient on enzyme replacement therapy. Comments on the dynamics of persistent storage process

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