Abstract
Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP. The two GAP forms in placental extracts were resolved by molecular sieve chromatography and appeared to have a monomeric native structure. Human recombinant type I GAP was produced intracellularly in Sf9 insect cells using a baculovirus expression vector, and 10-mg quantities were purified to homogeneity in three steps. Comparison of the purified native and recombinant GAP molecules revealed that all three displayed similar biological specific activities in an in vitro GAP assay. A polyclonal antibody to purified recombinant GAP was prepared and shown to neutralize the activity of both native and recombinant GAPs. The antibody was also highly specific for the detection of native GAP by Western blot. Type I and II GAP species were detected in approximately equal amounts in cytoplasmic extracts of human placenta, but only type I GAP was observed when other human tissues were examined.
Highlights
Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p2 1 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras
One neutralizing unit is defined as the amount of antibody required to neutralize 50% of the activity of 2 units of GAP
Purification of Native GAP Species from Human PlucentaNative GAP was purified from the cytoplasmic fraction of homogenized human placenta using a four-step procedure involving ammonium sulfate fractionation and a series of ionexchange chromatography steps
Summary
GAP preparations were measured using either an immunoprecipitation assay or a phosphate-release assay. The pool of concentrated GAP protein (-50 ml) was divided in half, and each portion was loaded onto a Sephacryl 200 column (5 X 90 cm; Pharmacia LKB Biotechnology Inc.) equilibrated in 50 mM Tris (pH 8.5) containing 100 mM sodium chloride, 1 mM EDTA, 0.1 mM DTT, 200 /IM PMSF, 1 &g/ml leupeptin, and 1 rg/ml pepstatin A. Both columns were eluted overnight by gravity flow at 1.5 ml/min. 20 rg of protein from each cytoplasmic extract was subjected to Western blot analysis
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