Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells.

摘要: Abstract rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of ras-transformed cell phenotype. We have identified GTPase-activating (GAP) specific for rap plasma membranes isolated from differentiated HL60 cells. The GAP activity remained quantitatively associated with membrane following washes buffered 1 M LiCl containing 20 mM EDTA but was solubilized detergents Nonidet P-40 and deoxycholate. On basis size-exclusion chromatography, membrane-associated (rap GAPm) appeared distinct detected cytosolic fraction molecular sizes forms were estimated to be 36 54 A, respectively. GAPm purified near homogeneity by successive column chromatographies presence detergent. corresponded single polypeptide migrated mass approximately 88 kDa on SDS/polyacrylamide gels. inactive toward GTP-bound p21ras, rho, G25K, rac-1 did not stimulate dissociation guanine nucleotide rap.