Synthesis and separation of tritium-labeled intermediates of the shikimate pathway |
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Authors: | Richard I Christopherson John F Morrison |
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Institution: | Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra ACT, Australia |
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Abstract: | 5-3H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB3H4 and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem.15, 428–435). The 5-3H]shikimate has been converted enzymatically to 5-3H]chorismate and 5-3H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatograhic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel. |
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Keywords: | Author to whom all correspondence should be addressed: Department of Biochemistry John Curtin School of Medical Research Australian National University PO Box 334 Canberra City ACT 2601 Australia |
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