Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein.
The substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography. On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points. The major peak of the substrate was further purified. It was purified about 1,800-fold with a recovery of 2.2% by the above procedures. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000. The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein. A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol. GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP. Thus, the ADP-ribosylation substrate is a GTP-binding protein. This protein, designated Gb (b for botulinum), is widely distributed in various tissues. It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000311:Adrenal Glands / Q000032:analysis*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003014:Clostridium botulinum / Q000201:enzymology*; D003600:Cytosol / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009243:NAD / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013873:Thionucleotides / Q000378:metabolism; D014018:Tissue Distribution
D013873:Thionucleotides; D009243:NAD; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins