Add time:08/19/2019 Source:sciencedirect.com
Publisher SummaryThis chapter discusses the assay and purification procedure of Edeine (cas 11006-90-9) synthetases. In assay method, binding of the amino acyl to the enzyme complex can be measured only if radioactive edeine constituent amino acids are available. Formation of edeine A and B is measured by incorporation of radioactive glycine and spermidine into these two compounds or microbiological assay. Purification involves preparation of crude enzymes, DEAE-cellulose chromatography, and sephadex G-200 filtration. Further purification of polyenzymes is obtained by filtration through a column (1.8 X 70 cm) of Sephadex G-200 in buffer A. Polyenzyme I or polyenzyme II (about 30 rag) is applied to the column and eluted with buffer A. Fractions of 2.8 ml with a flow rate 5 ml/hr are collected. Proteins and ATP-32PPi exchange dependent on edeines constituent amino acids are measured.
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