A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein. |
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Authors: | Y Nemoto T Namba T Teru-uchi F Ushikubi N Morii S Narumiya |
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Institution: | Department of Pharmacology, Kyoto University Faculty of Medicine, Japan. |
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Abstract: | 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. |
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