Studies of Sulfate Utilization by Algae: 9. Fractionation of a Cell-free System from Chlorella into Two Activities Necessary for the Reduction of Adenosine 3'-Phosphate 5'-Phosphosulfate to Acid-Volatile Radioactivity.

Further properties of the enzymatic system obtained from Chlorella pyrenoidosa (Emerson strain 3) which reduces adenosine 3'-phosphate 5'-phosphosulfate-(35)S to acid-volatile radioactivity, when fortified with Mg(2+) and 2, 3-dimercaptopropan-1-ol as reductant, are described. Optimal concentrations of adenosine 3'-phosphate 5'-phosphosulfate-(35)S and Mg(2+) and the pH optimum have been determined. 2,3-Dimercaptopropan-1-ol can be replaced by dithiothreitol, mercaptoethanol, reduced glutathione, cysteine, and cysteamine. Treatment of the crude extracts with ammonium sulfate and alumina C-gamma gel yields two fractions, designated "S" and "A," which must be recombined to obtain acid-volatile radioactivity. Further fractionation of fraction S by ammonium sulfate gradient elution and diethylaminoethyl cellulose chromatography yields approximately a 50-fold increase in specific activity compared to that found in the crude extract. This material appears to contain an active component with a molecular weight estimated by agarose gel chromatography of about 330,000.