THE PURIFICATION OF A RAP1 GTPASE-ACTIVATING PROTEIN FROM BOVINE BRAIN CYTOSOL
- Author(s)
- Nice, EC; Fabri, L; Hammacher, A; Holden, J; Simpson, RJ; Burgess, AW;
- Details
- Publication Year 1992-01-25,Volume 267,Issue #3,Page 1546-1553
- Journal Title
- JOURNAL OF BIOLOGICAL CHEMISTRY
- Publication Type
- Journal Article
- Abstract
- Two GTPase-activating proteins (GAPs) have been detected in extracts from bovine brain: GAP-1, which is specific for the activation of ras GTPases, and GAP-3, which is specific for the activation of the rap1 GTPases. We present a strategy for the purification to homogeneity of a cytosolic form of GAP-3 from bovine brain. The 100,000 x g supernatant from homogenized brains was chromatographed sequentially on DEAE Fast Flow, green H-E4BD Sepharose, Bio-Gel A1.5, hydroxyapatite, and phenyl-Sepharose prior to high resolution separation on Mono Q HR 5/5, phenyl-Superose HR 5/5, Mono Q PC 1.6/5, and Superose 12 PC 3.2/30. This procedure resulted in an approximately 18,000-fold purification, yielding 50-mu-g of GAP-3 from 1.6 kg of tissue. Purified cytosolic GAP-3 migrated as a single band of apparent M(r) 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on gel filtration cytosolic GAP-3 chromatographed as a dimer with an apparent M(r) 92,000. Purified GAP-3 does not activate ras or rho GTPases and possesses no intrinsic GTPase activity. Amino acid sequence data indicated a proline-rich N terminus. The amino acid sequences of peptides generated by Staphylococcus aureus V8 digestion of reduced and pyridine-ethylated GAP-3 showed no similarity to the predicted primary structure of GAP-1 or any other proteins in the nucleic acid or protein data bases. By comparison with the data of Rubinfeld et al. (Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W. J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042), it appears that the membrane-associated (M(r) 85,000-95,000) and cytosolic forms of GAP-3 are derived from equivalent, or closely related, genes.
- Publisher
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
- Keywords
- PUTATIVE EFFECTOR DOMAIN; RAS-RELATED GENE; BINDING PROTEIN; TYROSINE PHOSPHORYLATION; MOLECULAR-CLONING; EXCHANGE-REACTION; RAS-P21 GTPASE; GROWTH-FACTOR; P21 GTPASE; GAP
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Creation Date: 1992-01-25 12:00:00