Abstract
This chapter discusses the assay and purification procedure of actinomycin lactonase. The enzyme catalyzes the conversion of a neutral molecule to an acidic compound. The assay is based upon the use of radioactive actinomycin. Both product and substrate are extracted from the reaction mixture with an organic solvent at an acid pH. The acidic product is then extracted into aqueous alkaline media, which is counted in a liquid scintillation spectrometer. The purification procedure involves preparation of crude extract; freeze thawing, ammonium sulfate fractionation, calcium phosphate gel chromatography, DEAE-cellulose column chromatography, and sephadex G-200 column chromatography. The known properties of the enzyme include pH optimum for activity on substrate, 7.8; temperature optimum 38°; K m , 11.4 μM; V max 0.267 μmole per hour per milligram of protein. Significant inhibition with 40 mM silver, cobalt, chromium, copper, iron, mercury, magnesium, sodium, nickel, or zinc is observed.
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