METABOLISM OF 3,4-DIHYDROXYBUTYL-1-PHOSPHONATE IN BACILLUS SUBTILIS.
Item
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Title
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METABOLISM OF 3,4-DIHYDROXYBUTYL-1-PHOSPHONATE IN BACILLUS SUBTILIS.
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Identifier
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AAI8119662
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identifier
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8119662
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Creator
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KLEIN, DAVID ANDREW.
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Contributor
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Dr. Burton E. Tropp
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Date
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1981
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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 phosphonate isostere of L-glycerol 3-phosphate, 3,4-dihydroxybutyl-1-phosphonate, (DHBP), was found to inhibit the growth of both the glycerol phosphate (168) and ribitol phosphate strain (W23) teichoic acid strains of Bacillus subtilis. The inhibition was bacteriostatic for the 168 strain, but bactericidal for strain W23. The growth inhibition could be prevented by inclusion of phosphate, glucose, or glycerol in the growth medium.;Both strains incorporated intact {lcub}3-('3)H{rcub}-3,4-dihydroxybutyl-1-phosphonate, ({lcub}('3)H{rcub}-DHBP({lcub}('3)H{rcub}-DHBP), into a polar lipid, a non-dialyzable fraction found in the aqueous phase of a phenol extract, and the cell wall.;Lipids labeled in vivo with {lcub}('3)H{rcub}-DHBP were fractionated on a DEAE-cellulose column with an ammonium acetate gradient. The structure of the major tritium-labeled lipid, Fraction IV, (89-95% of the labeled lipids) was determined by degradation with phospholipase C and alkaline phosphatase. Fraction IV was identified as the phosphonate analogue of phosphatidylglycerol phosphate, (1,2-di-acyl)-sn-glyceryl D-phosphoryloxy-3'-hydroxy-1-phosphonate.;Both strains of B. subtilis convalently incorporated DHBP into the walls in vivo. The 168 strain had 5.4 times as much DHBP as the W23 strain, 57 and 11 mmole DHBP/mole of wall hexosamine, respectively.;The route by which DHBP became incorporated into B. subtilis cell walls was determined by demonstrating that DHBP is a substrate for CDP-glycerol pyrophosphorylase, forming the phosphonic acid analogue of CDP-glycerol, CMP-DHBP. CMP-DHBP, in turn, can participate in an in vitro teichoic acid synthesizing assay. The means by which DHBP can replace glycerol 3-phosphate in teichoic acid synthesis was thus demonstrated.;For CDP-glycerol pyrophosphorylase, the apparent Km of DHBP is 36 mM S-DHBP and the apparent Ki is 6.5 mM S-DHBP. The natural substrate, L -glycerol 3-phosphate, had an apparent Km of .18 mM. The apparent Vmax was 1.8 mmole CMP-DHBP/min-mg which was only one-third the rate obtained for L-glycerol 3-phosphate. These values suggest that DHBP can inhibit because it may be actively transported to large intracellular concentrations.;Explanations for the differential effect of DHBP between the two strains of B. subtilis are offered.
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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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Biochemistry
