Purification of a novel ras GTPase-activating protein from rat brain.
GTPase-activating protein (GAP) and neurofibromin, a gene product of neurofibromatosis type I gene, have been identified as factors that stimulate GTPase activity of ras p21. We have previously suggested the presence of novel GAP activity that is immunologically distinguishable from GAP or neurofibromin in both the cytosolic and the particulate fractions of rat brain (Hattori, S., Maekawa, M., and Nakamura, S. (1992) Oncogene 7, 481-485). We have purified this novel GAP molecule from the cytosolic fraction of rat brain by more than 200,000-fold by five successive column chromatographies with a recovery of 6%. Apparent molecular mass of this molecule was estimated to be 100 kDa (p100GAPras). The same p100GAPras was purified from the particulate fraction after extraction with high salt. The activation of GTPase was observed with normal ras p21 but not with oncogenic ras p21, Rap1B/smg21B, or Ram25K. The dissociation constant of p100GAPras toward ras p21 estimated by competitive inhibition using ras p21 in complex with nonhydrolyzable analog of GTP was two times higher than that of neurofibromin and was lower than that of GAP by 2 orders of magnitude. These results clearly indicate that p100GAPras is a novel ras GAP molecule.
Maekawa
M
M
Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
Nakamura
S
S
Hattori
S
S
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D017886:Durapatite; D005260:Female; D020690:GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007106:Immune Sera; D007700:Kinetics; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D007106:Immune Sera; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D017886:Durapatite