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.
Highlights
A substrate protein for botulinum C 3 ADP-ribosyl- in their putative effector domains and alters their functions transferase (C3 exoenzyme) in human plateletswas 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
When platelet cytosol andmembranes were incubated with C 3 exoenzyme and [32P]NADand subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis and isoelectric focusing, they gave onlyone [32P]ADP-ribosylatedband on each electrophoresis that showed an M,. of 22,000 and a PI of 6.0
Paterson et al [16] further demonstrated that microinjection of ADP-ribosylated rhoA protein into Swiss 3T3 cells induces similar dissolution of actin filaments and morphological changes, whereas the reappearance of actin filament networks is induced in contact-inhibited 3T3 cells by injection of Val14-rhoAmutant protein, which has a reduced GTPaseactivityandremainsconstitutivelyintheGTPbound form. These results suggest that theADP-ribosylation substrate proteins, rho proteins, have a role in the assembly of actin filament networks as well as in cell dodecyl sulfate-polyacrylamide gel electrophoresis, adhesion to the substratum
Summary
Platelets were prepared either from the peripheral blood of healthy human donors for subcellular distribution study or from outdated human platelet concentrates for protein purification. Platelets were freed of contaminating blood cells by centrifugation at 120 X g for 15 min and thenpelleted by centrifugation at 2300 X g for 30 min. The platelet pellets were resuspended in 10 mM sodium phosphate, pH 7.0, 5 mM KCl, 135 mM NaCl, and 10 mM EDTA and washed three times by centrifugation. The washed pellets were suspended in 0.1 of the original volume of 5 mM Tris-HC1, pH 7.4, 5 mM EGTA, 1 mM benzamidine hydrochloride, and 0.43 mM phenylmethylsulfonyl fluoride and homogenized by 10 strokes in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 100,000X g for 30 min for separation into the membrane and cytosol
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