PubMed · 5041948
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
A D Bangham. 1972. Model membranes.. https://doi.org/10.1016/0009-3084(72)90069-2
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
An N-bromoacetyl electrophile attached to the 5'-phosphate group of a purine-rich oligonucleotide affords sequence-specific alkylation of duplex DNA (at 37 degrees C, pH 7.4) through the formation of a specific purine.purine.pyrimidine triple-helical complex. In a 645 bp restriction fragment containing three consecutive guanine bases adjacent to the 3'-end of an oligonucleotide binding site, the yield of single-strand cleavage after piperidine treatment is 80% at the guanine base directly adjacent to the binding site and 88% overall. In an 837 bp restriction fragment containing two adjacent inverted repeats of the third strand binding site and a single 3'-guanine base, yields of single-strand cleavage are 87% on each strand at the 3'-guanine base. Double-strand cleavage was obtained in 61% yield at a single site in a 6.6 kbp plasmid containing the 837 bp fragment. Extension of triple helix mediated DNA alkylation from the pyrimidine to purine motif formally extends the number of sites in duplex DNA that can be cleaved in a sequence-specific and nucleotide-specific manner in good yields.
Second-order rate constants for transfer of the beta-D-galactopyranosyl group from the galactosyl-enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q beta-galactosidases to anionic nucleophiles have been determined. The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1 s-1. By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constant kcat/Km for catalysis of cleavage of beta-D-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl-enzyme intermediate. These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to azide ion. We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461. The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme. However, the reaction of formate ion with the galactosylated E461G enzyme gives only D-galactose. These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl-enzyme intermediates. The relative reactivity of anionic nucleophiles toward the covalent galactosyl-enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water. This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents.
In this work, 400 and 600 MHz 1H HPLC-NMR spectroscopic methods were developed and applied to separate and identify the positional glucuronide isomers and anomers of the model nonsteroidal antiinflammatory drug, 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid, in whole human urine. The HPLC methods utilized either an isocratic system, comprising 30% acetonitrile in water at pH 2.5, or a gradient elution system increasing from 30% to 60% acetonitrile, in order to achieve improved separation of the 2-, 3-, and 4-O-acylglucuronide isomers from the faster eluting endogenous urinary metabolites. Directly coupled stop-flow 1H HPLC-NMR spectroscopic measurements were made at the retention times indicated by the UV-monitored chromatographic peaks. The glucuronide isomers were identified from the 1H NMR spectra on the basis of their chemical shifts and spin-spin coupling patterns. The elution order was 4-O-acyl-, 3-O-acyl-, and finally 2-O-acylglucuronide, with tR values of 10.04, 11.68, and 12.64 min, respectively. Although the alpha- and beta-anomers of each of the positional isomers could not be separated in these solvent systems, they could be identified in the individual 1H NMR spectra. This work shows for the first time that directly coupled HPLC-NMR spectroscopy can be used directly to isolate and characterize acyl-migrated isomers of drug glucuronides in whole urine. This approach will be of value in the study of glucuronide acyl migration reactions of nonsteroidal antiinflammatory drugs and other xenobiotic ester glucuronides in whole biofluids.