A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein.
A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.
Nemoto
Y
Y
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Namba
T
T
Teru-uchi
T
T
Ushikubi
F
F
Morii
N
N
Narumiya
S
S
D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D001905:Botulinum Toxins; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006882:Hydroxyapatites; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D020742:rhoA GTP-Binding Protein
D006882:Hydroxyapatites; D000246:Adenosine Diphosphate Ribose; D017886:Durapatite; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein