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The adenosine kinase cDNA in a pET-24b plasmid was the generous gift of Dr. Beverly S. Mitchell. The plasmid was transformed into BL21-(DE3) cells, and the protein expressed and purified as described by: Spychala, J.; Datta, N. S.; Takabayashi, K.; Datta, M.; Fox, I. H.; Gribbin, T.; Mitchell, B. S. Proc. Natl. Acad. Sci. 1996, 93, 1232-1237.
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0345369756
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note
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i, pH 6, as the mobile phase. Baseline separation between ATP and 3,4-anhydroATP was obtained. The analogue was purified again by HPLC to ensure no ATP contamination. The product was characterized by NMR spectroscopy and mass spectiometry.
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12
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0019205736
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The AdoMet analogue was synthesized with 3′,4′-anATP and methionine as substrates by modifications of the assay procedure described in the paper. The compound was purified by cation exchange chromatography and characterized by NMR spectroscopy and mass spectrometry
-
A strain, DM22pK8 of AdoMet synthetase was a generous gift of Dr. George D. Markham. The enzyme was expressed and purified as described by: Markham, G. D.; Hafner, E. W.; Tabor, C. W.; Tabor, H. J. Biol. Chem. 1980, 255, 9082-9092. The AdoMet analogue was synthesized with 3′,4′-anATP and methionine as substrates by modifications of the assay procedure described in the paper. The compound was purified by cation exchange chromatography and characterized by NMR spectroscopy and mass spectrometry.
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0344076363
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note
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A small feature at g = 2.08 (3130 G) is observed in all spectra. This is a component of an axially symmetric signal that appears upon prolonged incubation of the enzyme and is believed to be due to a side reaction off the catalytic pathway.
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