M. Ruengjitchatchawalya et al., PURIFICATION AND CHARACTERIZATION OF THE IM-2-BINDING PROTEIN FROM STREPTOMYCES SP STRAIN FRI-5, Journal of bacteriology, 177(3), 1995, pp. 551-557
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.