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Identity of 'acid' beta-glucosidase and glucocerebrosidase in human spleen.

ABSTRACT: 1. Glucocerebrosidase, in association with a membrane-bound ;acid' beta-glucosidase, was separated from a soluble ;neutral' beta-glucosidase that had no activity towards glucocerebroside as substrate. 2. Glucocerebrosidase, as well as ;acid' beta-glucosidase activity depended upon the association of factor P (a heat-stable, soluble, acidic glycoprotein) with factor C (a heat-labile membrane-bound protein). 3. Factor C was solubilized under certain conditions. 4. Solubilized factor C, as well as membrane-bound factor C, could be alternatively stimulated by sodium taurocholate to give both glucocerebrosidase and ;acid' beta-glucosidase activities. 5. Membrane-bound factor C reacted optimally with factor P whereas solubilized factor C was preferentially stimulated by taurocholate. 6. Factor P-dependent glucocerebrosidase activity differed in kinetic properties from the taurocholate-stimulated enzyme activity. 7. The results are discussed in the light of (a) identity of glucocerebrosidase and ;acid' beta-glucosidase, (b) application in clinical diagnosis, (c) physiological significance of the enzyme system, and (d) polygenic inheritance in adult Gaucher's disease.

SUBMITTER: Ho MW

PROVIDER: S-EPMC1166008 | biostudies-other | 1973 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundMutations in GBA1, which encodes the lysosome enzyme β-glucocerebrosidase (also referred to as acid β-glucosidase or GCase), are the most common genetic risk factor for Parkinson disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies.ObjectiveThis study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays.MethodsTwo mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme used to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays.ResultsThe characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells (hESCs) revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration.ConclusionsThe hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.

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Identity of 'acid' beta-glucosidase and glucocerebrosidase in human spleen.

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