BIOCHEMICAL AND IMMUNOLOGIC STUDIES OF ACID ALPHA-GLUCOSIDASE DEFICIENCY, A GENETICALLY HETEROGENEOUS, INHERITED NEUROMUSCULAR DISEASE (ISOZYMES, POMPE DISEASE, MONOCLONAL ANTIBODY).
Item
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Title
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BIOCHEMICAL AND IMMUNOLOGIC STUDIES OF ACID ALPHA-GLUCOSIDASE DEFICIENCY, A GENETICALLY HETEROGENEOUS, INHERITED NEUROMUSCULAR DISEASE (ISOZYMES, POMPE DISEASE, MONOCLONAL ANTIBODY).
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Identifier
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AAI8629711
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identifier
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8629711
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Creator
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LABADIE, GUNDULA ULLRICH.
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Contributor
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Kurt Hirschhorn
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Date
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1986
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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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Chemistry, Biochemistry
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Abstract
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The overall objectives of these studies were to characterize the normal and mutant acid (alpha)-glucosidase (GAA) enzymes, and to characterize the nature of the enzymatic defect(s) in the subtypes and variants of GAA deficiency.;Accomplishments include the development of a panel of monoclonal antibodies (Mc Abs) to placental GAA and the identification of a covalent, active site-directed inhibitor (CBE) of the human enzyme.;Rocket immunoelectrophoresis with polyclonal (PC) anti-placental GAA Abs demonstrated extensive heterogeneity among and within the subtypes of glycogenosis II. The residual, catalytically inactive enzyme in the CRM-positive infantile subtype was unique in that it had a reduced immunoreactivity and an abnormal pI. Further immunologic studies using MC Abs showed that MC Ab Sp2/53 recognized three enzyme forms in several infantile and adult subtypes previously classified as CRM-negative using PC Abs. GAA isozymes 1 and 2, purified by conventional chromatographic procedures, were resolved into four distinct, catalytically active electrophoretic forms by isoelectric focusing. The isolated enzyme forms had the same physical and kinetic properties as the conventionally purified isozymes. The subunit composition changed from predominantly 73 kDa to predominantly 67 kDa with increasing electronegativity. Isozyme 4, the most electropositive of the GAA isozymes, was immunologically, physically and kinetically identical to GAA isozyme 1. These enzyme forms contained no sialic acid residues, and little or no phosphate, indicating that the observed charge heterogeneity was most likely due to differences in the protein backbone of the enzyme.;Tryptic peptide maps of the major protein species identified in purified GAA preparations have been generated. Comparative studies showed the electroeluted 96 (precursor), 73 and 67 kDa (mature) denatured enzyme forms. A 20 kDa form, a glycoprotein which consistently co-purified with GAA, was shown to be unrelated. Finally, the amino acid composition and N-terminal amino acid sequence of the 67 and 73 kDa subunits of GAA isozyme 1 have been obtained. The composition of the subunits was virtually identical. The unblocked, mature 73 kDa enzyme provided an amino-terminal sequence of 16 residues, 50% of which were coded for by low redundancy codons. (Abstract shortened with permission of author.).
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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.
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Program
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Biomedical Sciences
