Purification of the phospholipase D of Corynebacterium pseudotuberculosis by recycling isoelectric focusing.

Recycling isoelectric focusing was investigated as a method for purification of phospholipase D (PLD) from cultures of Corynebacterium pseudotuberculosis. Supernatant fluids from cultures of equine isolate 155 in brain-heart infusion broth were dialyzed against distilled water, concentrated by lyophilization, and fractionated by preparative isoelectric focusing in free solution in a pH 3 to 13 gradient with 6M urea. Protein concentration, pH, and PLD activity of the 10 resulting fractions were determined. Two PLD activity assays were used: release of 14C choline from labeled sphingomyelin and synergistic hemolytic activity with Rhodococcus equi factors. Enzyme activity focused in 2 fractions at pH 8.5 to 9.8. The synergistic hemolytic assay was simple and rapid for detecting PLD in partially purified fractions. Electrophoretic examination of the fraction containing the highest concentration of PLD activity revealed protein bands at 14, 21, and 31.7 kD Mr, suggesting purification to near-homogeneity. Proteins from the 31.7-kD band were labeled by antibodies in serum from a goat with chronic C pseudotuberculosis infection.