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Purification, characterization, and western blot analysis of human GTPase-activating protein from native and
R Halenbeck1, W J Crosier, R Clark
1Department of Protein Chemistry, Cetus Corporation, Emeryville, California 94608.
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.
Insights
Human ras GTPase-activating protein (GAP) stimulates normal N-ras p21 but not oncogenic variants. Researchers purified two GAP forms from placenta and confirmed their activity and specificity using recombinant versions and antibodies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Ras GTPase-activating protein (GAP) is crucial for regulating ras p21 activity.
- GAP's differential activity towards normal versus oncogenic N-ras p21 suggests a role in cancer biology.
- Understanding GAP's structure and function is key to deciphering ras signaling pathways.
Purpose of the Study:
- To purify and characterize different forms of human ras GAP.
- To compare the biological activity of native and recombinant GAP.
- To develop specific antibodies for GAP detection and functional studies.
Main Methods:
- Purification of native ras GAP from human placental tissue using chromatography.
- Production and purification of human recombinant type I GAP via baculovirus expression.
- In vitro GAP assay to measure GTPase-stimulating activity.
- Western blot analysis and antibody neutralization assays for specificity and activity confirmation.
Main Results:
- Two native ras GAP forms (Type I, Mr = 120,000, and Type II, Mr = 95,000) were purified from human placenta.
- Both native and recombinant GAP molecules exhibited similar specific activities in vitro.
- A polyclonal antibody effectively neutralized GAP activity and specifically detected native GAP.
- Type I and II GAP were found in equal amounts in placenta, but only Type I in other tissues.
Conclusions:
- Human placental extracts contain distinct Type I and Type II ras GAP molecules with conserved biological activities.
- Recombinant GAP production facilitates detailed functional and structural studies.
- The developed antibody is a valuable tool for specific detection and functional assessment of ras GAP.
- Tissue-specific expression patterns of GAP isoforms warrant further investigation into their distinct roles.