Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP.
Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins. We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c. Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity. Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained. The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae. These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1.
Garrett
M D
MD
Institute of Cancer Research, Chester Beatty Laboratories, London, U.K.
Major
G N
GN
Totty
N
N
Hall
A
A
D000595:Amino Acid Sequence; D002845:Chromatography; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen; D020703:ras GTPase-Activating Proteins; D020743:rhoB GTP-Binding Protein
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; C065725:IRA1 protein, S cerevisiae; D008565:Membrane Proteins; D011506:Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein