PURIFICATION OF A PLASMA MEMBRANE-ASSOCIATED GTPASE-ACTIVATING PROTEIN-SPECIFIC FOR RAP1/KREV-1 FROM HL60 CELLS

被引:93
作者
POLAKIS, PG
RUBINFELD, B
EVANS, T
MCCORMICK, F
机构
[1] CETUS CORP,DEPT PATHOL PHYSIOL,EMERYVILLE,CA 94608
[2] UNIV CALGARY,DEPT PHARMACOL & THERAPEUT,CELL REGULAT GRP,CALGARY T2N 4N1,ALBERTA,CANADA
关键词
D O I
10.1073/pnas.88.1.239
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differential HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis site-exclusion chromatography, the membrane-associated rap GAP (rap GApm) appeared from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 angstrom, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single poly-peptide that migrated with a molecular mass of almost-equal-to 88 kDa on SDA/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.
引用
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页码:239 / 243
页数:5
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