Purification of GTPase-activating protein specific for the rho gene products.
A GTPase-activating protein specific for the rho gene products (rho-GAP) was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of phenyl-Sepharose and CM-Sepharose, gel filtration on a TSK-gel G3000SW, and Mono S fast protein liquid chromatography. By these procedures the activity was purified about 36,000-fold with a recovery of 0.6%. The final preparation showed a major protein band at Mr 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stimulated GTP hydrolysis by the purified rho A protein in a time- and dose-dependent manner. No stimulation was found for ras p21. The ADP-ribosylation on the rho protein by botulinum C3 exoenzyme did not affect its interaction with the purified rho-GAP.
Morii
N
N
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Kawano
K
K
Sekine
A
A
Yamada
T
T
Narumiya
S
S
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000311:Adrenal Glands / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D020703:ras GTPase-Activating Proteins; D020743:rhoB GTP-Binding Protein
D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D000246:Adenosine Diphosphate Ribose; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020743:rhoB GTP-Binding Protein