Gaucher disease: Physical, kinetic and immunologic investigations of human and canine acid beta-glucosidase.
Item
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Title
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Gaucher disease: Physical, kinetic and immunologic investigations of human and canine acid beta-glucosidase.
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Identifier
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AAI8914745
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identifier
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8914745
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Creator
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Fabbro, Diane Elizabeth.
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Contributor
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Adviser: Gregory A. Grabowski
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Date
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1988
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics | Chemistry, Biochemistry
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Abstract
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Kinetic and immunologic techniques were developed to investigate the nature of the acid {dollar}\beta{dollar}-glucosidase ({dollar}\beta{dollar}-Glc) defects which result in human and canine Gaucher disease (GD). Two new affinity columns, using the potent inhibitors of {dollar}\beta{dollar}-Glc (N-alkyl-deoxynojirimycins) as affinity ligands, were synthesized and methods were developed to obtain homogeneous {dollar}\beta{dollar}-Glc from normal human placenta. Polyclonal and monoclonal (representing 14 different epitopes from 18 clones) antibodies were produced to the pure normal {dollar}\beta{dollar}-Glc. Monospecific polyclonal IgG and tritiated-bromo-conduritol B epoxide ( ({dollar}\sp3{dollar}H) Br-CBE), a specific covalent active site directed inhibitor of {dollar}\beta{dollar}-Glc, were used to quantitate the functional catalytic sites in normal and Type 1 Ashkenazi Jewish GD (AJGD) enzyme preparations: The k{dollar}\sb{lcub}\rm cat{rcub}{dollar} values for several new substrates with the mutant enzymes from spleen were about 1.5-fold less than the respective normal enzyme, indicating a nearly normal catalytic capacity of the mutant enzymes. Immunoblotting studies with polyclonal or several monoclonal antibodies indicated three molecular forms of {dollar}\beta{dollar}-Glc (M{dollar}\sb{lcub}\rm r{rcub}{dollar} = 67,000, 62,000 to 65,000 and 58,000) in fibroblast extracts from normals and Type 1 AJGD patients. In comparison, only one form of cross-reacting immunologic material (CRIM) was detected in fibroblast extracts from Types 2 and 3 or several non-Jewish Type 1 GD patients. These single CRIM forms were of variable M{dollar}\sb{lcub}\rm r{rcub}{dollar} in the different patients. Deglycosylation of the {dollar}\beta{dollar}-Glc in these fibroblast extracts resulted in a single CRIM form (M{dollar}\sb{lcub}\rm r{rcub}{dollar} = 56,000) in all cases, indicating that the core {dollar}\beta{dollar}-Glc polypeptide from all GD patients had a normal M{dollar}\sb{lcub}\rm r{rcub}{dollar} as assessed by SDS-PAGE. The CRIM molecular forms in the normal and GD sources were shown to bind ({dollar}\sp3{dollar}H) Br-CBE, i.e., they were {dollar}\beta{dollar}-Glc. Four monoclonal antibodies were shown to inhibit {dollar}\beta{dollar}-Glc activity by three different mechanisms: (1) blocking of the active site, (2) induction of a conformational change at the active site, or (3) both of these mechanisms. One of these monoclonal antibodies, MCAb61, proved useful for the delineation of the variants of GD based on the degree of its inhibition of the residual enzymatic activity. Studies of the canine GD indicated that it was an analogue of the neuronopathic human GD with normal levels of CRIM and thermolabile {dollar}\beta{dollar}-Glc activity. These studies indicate that human GD is highly heterogeneous at the phenotypic as well as the biochemical levels. The results also support the concept that all subtypes and variants of GD result from single base substitutions in the {dollar}\beta{dollar}-Glc structural gene which lead to a variety of processing abnormalities as well as a uniquely altered active site in Type 1 AJGD. Based on these studies, it is proposed that the allelic defects in the subtypes and variants of GD result from a dislocalization and/or an in vivo instability of {dollar}\beta{dollar}-Glc.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
