Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product.
The 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 was purified from porcine brain cytosol by acetone precipitation, CM-Sephadex, octyl-Sepharose and TSK phenyl-5PW HPLC chromatography to apparent homogeneity. ADP-ribosylation of the protein was increased by guanine nucleotides (GTP, GDP, GTP gamma S, each 100 microM) but not by GMP, ATP or ATP gamma S. The purified 22 kDa protein bound maximally 0.9 mol [35S]GTP gamma S and hydrolyzed GTP with the rate 0.007 mol per mol protein. Amino acid sequences were obtained from two tryptic peptides, selected from an in situ digestion of Immobilon electrotransferred, gel purified ADP-ribosylated substrate. The two sequences obtained, cover 23 residues from the corresponding sequences in human rho.
Braun
U
U
Rudolf-Buchheim-Institut für Pharmakologie, Universität Giessen, FRG.
Habermann
B
B
Just
I
I
Aktories
K
K
Vandekerckhove
J
J
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D013873:Thionucleotides / Q000378:metabolism; D020743:rhoB GTP-Binding Protein
D008565:Membrane Proteins; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D036002:ADP Ribose Transferases; D001905:Botulinum Toxins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein