Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources.
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.
Halenbeck
R
R
Department of Protein Chemistry, Cetus Corporation, Emeryville, California 94608.
Crosier
W J
WJ
Clark
R
R
McCormick
F
F
Koths
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D019204:GTP-Binding Proteins