An examination of the relationship between the structure and topology of peptidoglycan synthesis in bacterial membranes and enzyme activity.
Item
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Title
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An examination of the relationship between the structure and topology of peptidoglycan synthesis in bacterial membranes and enzyme activity.
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Identifier
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AAI9218278
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identifier
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9218278
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Creator
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Talbot, Maureen Kelly.
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Contributor
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Adviser: Frank Margolis
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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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Chemistry, Biochemistry | Health Sciences, Oncology | Biology, Microbiology
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Abstract
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The peptidoglycan biosynthetic pathway proceeds from nucleotide-linked precursors in the cytoplasm, through lipid-linked intermediates associated with the cytoplasmic membrane, to an insoluble, cross-linked, polymeric product outside the membrane. In this work, right-side out (RSO) and inside-out (ISO) E. coli membrane vesicles were used to study the membrane-associated steps. Vesicles of both orientations incorporated label from UDP- ({dollar}\sp{14}{dollar}C) N-acetylglucosamine (UDP- ({dollar}\sp{14}{dollar}C) GlcNAc) in the presence of UDP-N-acetylmuramyl-L-ala-D-glu-meso-DAP-D-ala-D-ala (UDP-MurNAc-pentapeptide), and from UDP-MurNAc- ({dollar}\sp3{dollar}H) pentapeptide in the presence and absence of UDP-GlcNAc. The activity seen in RSOs was surprising because the synthetic enzymes, phospho-N-acetylmuramyl-pentapeptide transferase (translocase) and N-acetylglucosamine transferase, were presumed to be localized on the inner surface of the cytoplasmic membrane. Since the phosphorylated substrates cannot cross the membrane, the enzymes measured must be accessible from both sides of the membrane. Differential effects of several proteases eliminated the possibility that the enzymes are randomly oriented in the membrane. In addition, EDTA-treated and whole, untreated E. coli cells were shown to incorporate label from the nucleotide-linked precursors into SDS-insoluble peptidoglycan.;Inhibitors of specific steps of peptidoglycan synthesis revealed that, in addition to the translocase and the N-acetylglucosamine transferase, the undecaprenyl pyrophosphate phosphatase and at least the transglycosylase domain of the penicillin-binding proteins are active in vesicles of both orientations.;The products of incorporation of UDP-MurNAc- ({dollar}\sp3{dollar}H) pentapeptide and UDP- ({dollar}\sp{14}{dollar}C) GlcNAc by RSOs and ISOs was examined by TLC. Vesicles of both orientations synthesized a rapidly migrating labelled material that comigrated with authentic ({dollar}\sp{14}{dollar}C) -labelled lipid-linked disaccharide on TLC. A significant amount of polymer was produced by ISOs; RSOs produced very little of this product.;A model of a membrane-spanning multi-enzyme complex is proposed to correlate the activity in vesicles of both orientations with the physical and biochemical observations made in other laboratories.
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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.
