The molecular and biochemical analysis of alpha-galactosidase A activity variants.
Item
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Title
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The molecular and biochemical analysis of alpha-galactosidase A activity variants.
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Identifier
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AAI9224812
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identifier
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9224812
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Creator
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Fitzmaurice, Thomas Francis.
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Contributor
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Adviser: Ann Henderson
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Date
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1992
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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, Molecular | Chemistry, Biochemistry
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Abstract
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Biochemical and molecular studies were carried out on the lysosomal hydrolase {dollar}\alpha{dollar}-galactosidase A (E.C. 3.2.1.22, {dollar}\alpha{dollar}-Gal A) in order to characterize variant enzyme activity. Determination of thermal stability, specific activity and enzyme kinetics was carried out on {dollar}\alpha{dollar}-Gal A that had been partially purified from the lymphoblasts of an individual with residual enzyme activity as well as on enzyme from a high-activity variant exhibiting plasma enzyme activity levels of approximately 3 times the normal mean.;Characterization of {dollar}\alpha{dollar}-Gal A from a residual activity variant revealed an enzyme with reduced thermostability and altered kinetic properties. This enzyme had half-lives of 11 and 8 minutes at 41{dollar}\sp\circ{dollar}C and pH 4.6 or 7.4, respectively, as compared to normal control values of 33 and 44 minutes under the same conditions. The K{dollar}\sb{lcub}\rm m{rcub}{dollar} of the residual activity enzyme for 4-methylumbelliferyl-{dollar}\alpha{dollar}-D-galactopyranoside (4-MU-{dollar}\alpha{dollar}-Gal) was 4.1 mM as compared to 3.0 mM for the normal enzyme. The enzyme was also shown to have reduced affinity for the potent inhibitor, D-galacto-deoxynojirimycin, giving further evidence that the active site of the residual activity enzyme was altered. Molecular analysis of the residual activity allele revealed an A to G transition in exon 6 of the {dollar}\alpha{dollar}-Gal A cDNA that resulted in the substitution of valine for methionine at residue 296 of the resulting protein (M296V).;Characterization of enzyme from the high-activity variant determined that its kinetic and physical properties were the same as those of normal enzyme. {dollar}\alpha{dollar}-Gal A messenger RNA levels in the high-activity variant were also shown to be normal, indicating absence of increased transcription or stability. Sequence analysis of the high-activity allele revealed a G to A transition 30 nucleotides 5{dollar}\sp\prime{dollar} of the translation start site in the cDNA. This mutation was inherited in an X-linked manner and also found in three additional, unrelated high-activity individuals thereby demonstrating its causative nature. In vitro transcription and translation of the normal and high-activity alleles resulted in normal levels of {dollar}\alpha{dollar}-Gal A protein suggesting that some form of cell-specific regulation mechanism may be responsible for the increased amount of enzyme activity seen in vivo.
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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.
