Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0155339 (Brown)
 12,436 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp. 212 Dec 70

Ras GTPase activating proteins (GAPs) contain an invariant motif, -FLR-, within the most conserved region of their catalytic domains. Certain mutations in this motif have greatly reduced activity (Skinner, R. H., Bradley, S., Brown, A. L., Johnson, N. J., Rhodes, S., Stammers, D. K., and Lowe, P. N. (1991) J. Biol. Chem. 266, 14163-14166), but it was not determined whether the reduced activity was due to loss of binding or impaired catalysis. In order to address this question, we have developed a simple physical method to study formation of GAP.p21ras complexes. This utilizes the increase of fluorescence anisotropy upon binding of GAP to p21ras complexed with 2'(3')-O-(N-methylanthraniloyl) (mant) derivatives of guanine nucleotides. Dissociation constants obtained for the catalytic domains of either p120-GAP (GAP-344) or neurofibromin (NF1-GRD) with normal and Leu-61 p21ras proteins are comparable with those obtained by kinetic methods. In the course of these studies, we found, in contrast to previous observations, that both GAP and NF1-GRD can weakly activate the GTPase of Leu-61 mutant p21, showing that Gln-61 is not absolutely required for the stimulation of GTPase activity by GAPs. The fluorescence anisotropy method allowed us to show that mutation of Arg-903, within the FLR motif of GAP, can result in protein defective in catalysis but not in binding to p21ras. These data suggest a direct role for this residue in catalyzing GTP hydrolysis on p21ras, possibly by contributing a catalytic group to the p21 active site. This method is independent of the catalytic activity of the proteins, and so it could be extended generally to the measurement of binding of effector molecules, exchange factors, or other macromolecules to guanine nucleotide-binding proteins.
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PMID:Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method. Essential role of Arg-903 of GAP in activation of GTP hydrolysis on p21ras. 849 56

Ras proteins are small GTP-binding proteins which are critical for cell signaling and proliferation. Four Ras isoforms exist: Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B. The carboxyl termini of all four isoforms are post-translationally modified by farnesyl protein transferase (FPT). Prenylation is required for oncogenic Ras to transform cells. Recently, it was reported that Ki-Ras4B is also an in vitro substrate for the related enzyme geranylgeranyl protein transferase-1 (GGPT-1) (James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 6221-6226). In the current studies, we compared the four isoforms of Ras as substrates for FPT and GGPT-1. The affinity of FPT for Ki-Ras4B (Km = 30 nM) is 10-20-fold higher than that for the other Ras isoforms. Consistent with this, when the different Ras isoforms are tested at equimolar concentrations, it requires 10-20-fold higher levels of CAAX-competitive compounds to inhibit Ki-Ras4B farnesylation. Additionally, we found that, as reported for Ki-Ras4B, N-Ras and Ki-Ras4A are also in vitro substrates for GGPT-1. Of the Ras isoforms, N-Ras is the highest affinity substrate for GGPT-1 and is similar in affinity to a standard GGPT-1 substrate terminating in leucine. However, the catalytic efficiencies of these geranylgeranylation reactions are between 15- and 140-fold lower than the corresponding farnesylation reactions, largely reflecting differences in affinity. Carboxyl-terminal peptides account for many of the properties of the Ras proteins. One interesting exception is that, unlike the full-length N-Ras protein, a carboxyl-terminal N-Ras peptide is not a GGPT-1 substrate, raising the possibility that upstream sequences in this protein may play a role in its recognition by GGPT-1. Studies with various carboxyl-terminal peptides from Ki-Ras4B suggest that both the carboxyl-terminal methionine and the upstream polylysine region are important determinants for geranylgeranylation. Furthermore, it was found that full-length Ki-Ras4B, but not other Ras isoforms, can be geranylgeranylated in vitro by FPT. These findings suggest that the different distribution of Ras isoforms and the ability of cells to alternatively process these proteins may explain in part the resistance of some cell lines to FPT inhibitors.
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PMID:Characterization of Ha-ras, N-ras, Ki-Ras4A, and Ki-Ras4B as in vitro substrates for farnesyl protein transferase and geranylgeranyl protein transferase type I. 909 72