UTILIZATION OF CIBACRON BLUE 3G-A SEPHAROSE 6B IN THE ISOLATION AND ENRICHMENT OF PYRUVATE, PHOSPHATE DIKINASE, ALANINE DEHYDROGENASE, AND PHOSPHOENOLPYRUVATE CARBOXYLASE FROM MITOMYCIN-PRODUCING STREPTOMYCES VERTICILLATUS

Pyruvate, phosphate dikinase (E.C. 2.7.9.1) has been demonstrated in mitomycin-producing Streptomyces verticillatus. Low levels of activity are detectable in noninduced cultures; however, in alanine or pyruvate containing media the enzyme activity is induced 20 to 40 fold. Assays for the dikinase are subject to very large interferences if pyruvate kinase and adenylate kinase are present together. or if alanine dehydrogenase is present. Chromatography of dialyzed cell-free extracts of induced S. verticillatus on Cibacron Blue 3G-A Sepharose 6B affords a clean separation of the dikinase from these interfering enzymes which are present in this organism. While the dikinase is eluted in the early fractions, pyruvate kinase and alanine deh drogenase are retained on the column. The dikinase is inhibited by free Cibacron 3G-A Blue and therefore its non-binding to the dye Sepharose column is presently unexplained. Dikinase activity has been detected in two of four strains of Streptomyces tested and it was found to be absent in rifamycin-producing Nocardia mediterranei. Lactate dehydrogenase (E.C. 1.1.1.27) activity could not be detected in S. verticillatus, but alanine dehydrogenase (E.C. 1.4.1.1) has been purified 75 fold in a single step by elution with 0.25 mM NADH from Cibacron Blue 3G-A Sepharose 6B. Further studies have shown the presence of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) in extracts of S. verticillatus. This acetyl coenzyme A activated enzyme readily binds to the dye columns and can be specifically eluted with acetyl-coenzyme A. It presumably interacts with the dye on the column by way of its allosteric site.