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.

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Abstract
Pyruvate, phosphate dikinase (E.C. 2.7.9.1) was demonstrated in mitomycin-producing S. verticillatus. Low levels of activity are detectable in noninduced cultures; 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 dehydrogenase are retained on the column. The dikinase is inhibited by free Cibacron 3G-A Blue and its non-binding to the dye Sepharose column is presently unexplained. Dikinase activity was detected in 2 of 4 strains of Streptomyces tested and was 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) was purified 75-fold in a single step by elution with 0.25 mM NADH from Cibacron Blue 3G-A Sepharose 6B. Further studies showed the presence of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) in extracts of S. verticillatus. This acetyl Co A activated enzyme readily binds to the dye columns and can be specifically eluted with acetyl-Co A. It presumably interacts with the dye on the column by way of its allosteric site.