PURIFICATION AND CHARACTERIZATION OF THE IM-2-BINDING PROTEIN FROM STREPTOMYCES SP STRAIN FRI-5

IM-2 )-2-(1'-hydroxybutyl)-3-(hydroxymethyl)butanolide] of Streptomyce s sp. strain FRI-5 is one of the butyrolactone autoregulators of Strep tomyces species and triggers production of blue pigment as well as the nucleoside antibiotics showdomycin and minimycin. A tritium-labeled I M-2 analogue, oxy-[4',5'-H-3]pentyl)-3-(hydroxymethyl)butanolide ([H-3 ]IM-2-C-5; 40 Ci/mmol), was synthesized for a competitive binding assa y, and an IM-2-specific binding protein was found to be present in the crude cell extract of Streptomyces sp. strain FRI-5. During cultivati on for 24 h, the specific IM-2-binding activity increased rapidly, rea ched a plateau at 10 to 14 h, and declined sharply thereafter, showing only 6% activity after 24 h of cultivation, A Scatchard plot of the b inding data demonstrated that the dissociation constant (K-d for [H-3] IM-2-C-5 was 1.3 nM, while the K-d for a H-3-labeled virginiae butanol ide (VB) analogue, oxy-[6',7'-H-3]heptyl)-3-(hydroxymethyl)butanolide ([H-3]VB-C-7), another butyrolactone autoregulator possessing the oppo site configuration at C-1' was 35 nM. Furthermore, at a 15-fold molar excess, the effectiveness of several autoregulators as nonlabeled comp etitive ligands against [H-3]IM-2-C-5 was IM-2 type > VB-C type >> A-f actor type, indicating that the binding protein in Streptomyces sp. st rain FRI-5 is highly specific toward IM-2. Ultracentrifugation showed that the IM-2-binding protein is present almost exclusively in the 100 ,000 x g supernatant fraction, indicating that the binding protein is a cytoplasmic soluble protein. The binding protein was purified by amm onium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S -100 HR gel filtration, DEAE-5PW high-performance liquid chromatograph y (HPLC), and phenyl-5PW HPLC. The apparent M(r) of the native IM-2-bi nding protein as determined by molecular sieve HPLC was about 60,000 i n the presence of 0.5, 0.3, or 0.1 M KCI, while by sodium dodecyl sulf ate-polyacrylamide gel electrophoresis it was about 27,000, suggesting that the native binding protein is present in the form of a dimer.