Purification and characterization of virginiae butanolide C-binding protein, a possible pleiotropic signal-transducer in Streptomyces virginiae.
Virginiae butanolide C (VB-C) is an autoregulator which triggers virginiamycin production in Streptomyces virginiae. A new binding assay with tritium-labeled VB-C analogue (2,3-cis-2-(1'-hydroxy-[6',7'-3H]heptyl)-3-(hydroxymethyl)butanolide+  ++ ) was developed and a specific VB-C binding protein was purified to homogeneity from crude extracts of S. virginiae by ammonium sulfate fractionation, DEAE-Sephacel and Sephadex G-100 column chromatographies, hydrophobic HPLC on phenyl 5PW and native polyacrylamide gel electrophoresis. The VB-C binding protein showed an apparent Mr of 35,800 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Mr of 26,000 approximately 44,000 on native molecular sieve HPLC, indicating the monomeric nature of the binding protein. The binding protein efficiently bound to a VB affinity column and eluted specifically by VB-C, which confirmed the specific nature of the binding protein. The binding activity decreased by 40% in the presence of genomic DNA from S. virginiae, indicating interaction between the VB-C binding protein and the DNA.
Kim
H S
HS
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Tada
H
H
Nihira
T
T
Yamada
Y
Y
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification*; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004269:DNA, Bacterial / Q000494:pharmacology; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005663:Furans / Q000378:metabolism*; D008024:Ligands; D015394:Molecular Structure; D015398:Signal Transduction; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D004269:DNA, Bacterial; D005663:Furans; D008024:Ligands; C052546:virginiamycin butanolide C; D015107:4-Butyrolactone