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

被引:26
作者
RUENGJITCHATCHAWALYA, M [1 ]
NIHIRA, T [1 ]
YAMADA, Y [1 ]
机构
[1] OSAKA UNIV,FAC ENGN,DEPT BIOTECHNOL,SUITA,OSAKA 565,JAPAN
关键词
D O I
10.1128/jb.177.3.551-557.1995
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
IM-2 [(2R,3R,1'R)-2-(1'-hydroxybutyl)-3-(hydroxymethyl)butanolide] of Streptomyces sp. strain FRI-5 is one of the butyrolactone autoregulators of Streptomyces species and triggers production of blue pigment as well as the nucleoside antibiotics showdomycin and minimycin. A tritium-labeled IM-2 analogue, 2,3-trans-2(1'-beta-hydroxy-[4',5'-H-3]pentyl)-3-(hydroxymethyl)butanolide ([H-3]IM-2-C-5; 40 Ci/mmol), was synthesized for a competitive binding assay, and an IM-2-specific binding protein was found to be present in the crude cell extract of Streptomyces sp. strain FRI-5. During cultivation for 24 h, the specific IM-2-binding activity increased rapidly, reached 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 binding 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 butanolide (VB) analogue, 2-(1'-alpha-hydroxy-[6',7'-H-3]heptyl)-3-(hydroxymethyl)butanolide ([H-3]VB-C-7), another butyrolactone autoregulator possessing the opposite configuration at C-1' was 35 nM. Furthermore, at a 15-fold molar excess, the effectiveness of several autoregulators as nonlabeled competitive ligands against [H-3]IM-2-C-5 was IM-2 type > VB-C type >> A-factor type, indicating that the binding protein in Streptomyces sp. strain 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 ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-100 HR gel filtration, DEAE-5PW high-performance liquid chromatography (HPLC), and phenyl-5PW HPLC. The apparent M(r) of the native IM-2-binding protein as determined by molecular sieve HPLC was about 60,000 in the presence of 0.5, 0.3, or 0.1 M KCI, while by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was about 27,000, suggesting that the native binding protein is present in the form of a dimer.
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页码:551 / 557
页数:7
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