Abstract
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'-3H]pentyl)-3-(hydroxymethyl)butano lide ([3H]IM-2-C5; 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 (Kd) for [3H]IM-2-C5 was 1.3 nM, while the Kd for a 3H-labeled virginiae butanolide (VB) analogue, 2-(1'-alpha-hydroxy-[6',7'-3H]heptyl)-3-(hydroxymethyl)butanolide ([3H]VB-C7), 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 [3H]IM-2-C5 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 Mr 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 KCl, 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.
Full Text
The Full Text of this article is available as a PDF (300.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Gräfe U., Schade W., Eritt I., Fleck W. F., Radics L. A new inducer of anthracycline biosynthesis from Streptomyces viridochromogenes. J Antibiot (Tokyo) 1982 Dec;35(12):1722–1723. doi: 10.7164/antibiotics.35.1722. [DOI] [PubMed] [Google Scholar]
- Kim H. S., Nihira T., Tada H., Yanagimoto M., Yamada Y. Identification of binding protein of virginiae butanolide C, an autoregulator in virginiamycin production, from Streptomyces virginiae. J Antibiot (Tokyo) 1989 May;42(5):769–778. doi: 10.7164/antibiotics.42.769. [DOI] [PubMed] [Google Scholar]
- Kim H. S., Tada H., Nihira T., Yamada Y. Purification and characterization of virginiae butanolide C-binding protein, a possible pleiotropic signal-transducer in Streptomyces virginiae. J Antibiot (Tokyo) 1990 Jun;43(6):692–706. doi: 10.7164/antibiotics.43.692. [DOI] [PubMed] [Google Scholar]
- Kondo K., Higuchi Y., Sakuda S., Nihira T., Yamada Y. New virginiae butanolides from Streptomyces virginiae. J Antibiot (Tokyo) 1989 Dec;42(12):1873–1876. doi: 10.7164/antibiotics.42.1873. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Miyake K., Horinouchi S., Yoshida M., Chiba N., Mori K., Nogawa N., Morikawa N., Beppu T. Detection and properties of A-factor-binding protein from Streptomyces griseus. J Bacteriol. 1989 Aug;171(8):4298–4302. doi: 10.1128/jb.171.8.4298-4302.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nihira T., Shimizu Y., Kim H. S., Yamada Y. Structure-activity relationships of virginiae butanolide C, an inducer of virginiamycin production in Streptomyces virginiae. J Antibiot (Tokyo) 1988 Dec;41(12):1828–1837. doi: 10.7164/antibiotics.41.1828. [DOI] [PubMed] [Google Scholar]
- Yamada Y., Sugamura K., Kondo K., Yanagimoto M., Okada H. The structure of inducing factors for virginiamycin production in Streptomyces virginiae. J Antibiot (Tokyo) 1987 Apr;40(4):496–504. doi: 10.7164/antibiotics.40.496. [DOI] [PubMed] [Google Scholar]