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1.
Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s, ras p21-like GTP-binding proteins 总被引:16,自引:0,他引:16
T Yamamoto K Kaibuchi T Mizuno M Hiroyoshi H Shirataki Y Takai 《The Journal of biological chemistry》1990,265(27):16626-16634
Novel regulatory proteins for smg p21A and -B, ras p21-like GTP-binding proteins (G proteins) having the same putative effector domain as ras p21s, were purified to near homogeneity from bovine brain cytosol and characterized. These regulatory proteins, designated as GDP dissociation stimulator (GDS) 1 and -2, stimulated the dissociation of both [3H]GDP and [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) from smg p21s to the same extent. smg p21 GDS1 and -2 also stimulated the binding of [35S]GTP gamma S to the GDP-bound form of smg p21s but not that to the guanine nucleotide-free form. These actions of smg p21 GDS1 and -2 were specific for smg p21s and inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, rhoB p20, and smg p25A. Neither smg p21 GDS1 nor -2 stimulated the GTPase activity of smg p21s and by itself showed [35S]GTP gamma S-binding or GTPase activity. smg p21 GDS1 and -2 showed very similar physical and kinetic properties and were indistinguishable by peptide map analysis. The Mr values of smg p21 GDS1 and -2 were estimated to be about 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S values, indicating that smg p21 GDS1 and -2 are composed of a single polypeptide without a subunit structure. smg p21 GDS1 and -2 were distinguishable from GTPase activating proteins (GAPs) for the ras and rho proteins, and smg p21B, and GDP dissociation inhibitors for smg p25A and the rho proteins previously identified in bovine brain cytosol. These results indicate that bovine brain contains regulatory proteins for smg p21s that stimulate the dissociation of GDP from and thereby the subsequent binding of GTP to smg p21s in addition to smg p21 GAP. It is likely that the conversion from the GDP-bound inactive form of smg p21s to the GTP-bound active form is regulated by smg p21 GDS and that its reverse reaction is regulated by smg p21 GAP. 相似文献
2.
The product of the rap2 gene, member of the ras superfamily. Biochemical characterization and site-directed mutagenesis 总被引:2,自引:0,他引:2
I Lerosey P Chardin J de Gunzburg A Tavitian 《The Journal of biological chemistry》1991,266(7):4315-4321
The human rap2 gene encodes a 183 amino acid protein that shares 46% identity with the K-ras p21. Its cDNA was engineered and inserted into the bacterial expression vector ptac; this allowed the production of high levels of soluble recombinant protein in Escherichia coli that was purified to near homogeneity. The rap2 protein binds GTP and exhibits a low intrinsic GTPase activity (rate constant of 0.5 x 10(-2) min-1). It exchanges its bound GDP with a half-life of 18 min at 37 degrees C in the presence of 10 mM Mg2+. Under the same conditions, the dissociation of bound GTP was at least 25-fold slower showing that the rap2 protein has a much higher affinity for GTP than GDP. The contribution of individual domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 12 results in a 2-fold decrease in the GDP dissociation rate constant and GTPase activity. Replacement of the Ser at position 17 by Asn severely impairs the GTP binding ability of the protein and points to an important role of this residue in the coordination of Mg2+. Mutation of Thr-35 to Ala results in a decreased affinity for GTP and a reduction (3-fold) of the GTPase activity. Finally, substitution of Thr-145 by Ile leads to an imperfect binding of guanyl nucleotides as exemplified by an increase in their dissociation rate constants and reduction of the GTPase activity of the protein. These properties of the normal and mutant rap2 proteins are compared with those of ras p21 carrying similar substitutions and are discussed in relation to the structural models proposed for ras p21. 相似文献
3.
A novel method for the analysis of putative G-proteins has been developed that reveals the existence of a large family of GTP/GDP-binding proteins with similar characteristics to those of p21ras in 3T3 cell plasma membranes. In the presence of Mg2+, exchange of [alpha-32P]GDP with prebound ligand was very slow, but, as with p21ras, exchange was dramatically accelerated by excess EDTA. In the presence of Mg2+, three classes of binding sites were distinguishable. However, no p21ras was detected in the membranes with the pan-reactive anti-ras antibody, Y13-259. Gel filtration analysis resolved two peaks of binding activity centering at 60 and 21 kilodaltons. High resolution anion exchange chromatography separated at least 11 unique GDP-binding proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized membranes, none of which cross-reacted with a pan-G alpha antiserum. Analogous to p21ras, the binding activities of 9 of the 11 species were sensitive to the thiol reagent N-ethylmaleimide, and six peaks possessed detectable GTPase activity in the absence of extrinsic factors. Addition of cytosol activated the GTPase activity of four of the peaks. We infer that the 11 peaks represent novel, small molecular weight guanine nucleotide-binding proteins, similar to those recently described in brain membranes. 相似文献
4.
A Kikuchi T Sasaki S Araki Y Hata Y Takai 《The Journal of biological chemistry》1989,264(16):9133-9136
The smg-21 GTP-binding protein (smg p21) has the same effector domain as the ras proteins (ras p21s) and is identical with the proteins of the rap1A and Krev-1 genes. In this paper, two proteins stimulating the GTPase activity of smg p21 are partially purified from bovine brain cytosol. These proteins, designated as smg p21 GTPase-activating protein (GAP) 1 and 2, are separated from a c-ras p21 GAP described previously by column chromatographies. smg p21 GAP1 and -2 stimulate the GTPase activity of only smg p21 but not that of c-Ha-ras p21 or the rho and smg-25A GTP-binding proteins. smg p21 GAP1 or -2 does not stimulate the dissociation of guanosine 5'-3-O-(thio)triphosphate or GDP from smg p21. smg p21 GAP1 or -2 themselves do not have GTP/GDP binding or GTPase activity. The Mr values of smg p21 GAP1 and -2 are estimated to be 250-400 x 10(3) and 80-100 x 10(3) by gel filtration and sucrose density gradient ultracentrifugation, respectively. The activity of smg p21 GAP1 and -2 is killed by tryptic digestion or heat boiling. These results indicate that bovine brain contains two smg p21 GAPs in addition to c-ras p21 GAP. 相似文献
5.
A gene coding for the novel ras protein, p21X, in which the domains of guanine binding and phosphate binding were exchanged, was constructed and expressed in Escherichia coli. The gene product, p21X, showed GTP binding activity, but no GPTase activity. In addition, p21X revealed binding activity toward ATP and CTP. In a competitive binding assay, [3H]GTP binding to p21X was inhibited in the presence of ATP, CTP and UTP, ITP as well as GDP, GTP and dGTP. 相似文献
6.
Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s 总被引:17,自引:0,他引:17
Y Hata A Kikuchi T Sasaki M D Schaber J B Gibbs Y Takai 《The Journal of biological chemistry》1990,265(13):7104-7107
ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity. 相似文献
7.
N Ohga A Kikuchi T Ueda J Yamamoto Y Takai 《Biochemical and biophysical research communications》1989,163(3):1523-1533
A novel regulatory protein for rhoB p20, a ras p21-like GTP-binding protein (G protein), was partially purified from the cytosol fraction of rabbit intestine. This protein, designated as rhoB p20 GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP from rhoB p20. rhoB p20 GDI also inhibited the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. GDI did not affect the GTPase activity of rhoB p20 and by itself showed no GTP gamma S-binding activity. GDI was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p21 and smg p25A. The Mr value of GDI was estimated to be about 27,000 from the S value. These results indicate that rabbit intestine contains a novel regulatory protein that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to rhoB p20. 相似文献
8.
J D Graves J Downward S Rayter P Warne A L Tutt M Glennie D A Cantrell 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(11):3709-3712
T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism. 相似文献
9.
B Rubinfeld G Wong E Bekesi A Wood E Heimer F McCormick P Polakis 《International journal of peptide and protein research》1991,38(1):47-53
Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras. 相似文献
10.
Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. 总被引:16,自引:9,他引:16
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J Tucker G Sczakiel J Feuerstein J John R S Goody A Wittinghofer 《The EMBO journal》1986,5(6):1351-1358
v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin. 相似文献
11.
T Ueda A Kikuchi N Ohga J Yamamoto Y Takai 《The Journal of biological chemistry》1990,265(16):9373-9380
A novel regulatory protein for the rho proteins (rhoA p21 and rhoB p20), belonging to a ras p21/ras p21-like small molecular weight (Mr) GTP-binding protein (G protein) superfamily, was purified to near homogeneity from bovine brain cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor (GDI) for the rho proteins (rho GDI), inhibited the dissociation of GDP from rhoB p20 and the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. The Mr value of rho GDI was estimated to be about 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S value, indicating that rho GDI is composed of a single polypeptide without a subunit structure. The isoelectric point was about pH 5.7. rho GDI made a complex with the GDP-bound form of rhoB p20 with a molar ratio of 1:1 but not with the GTP gamma S-bound or guanine nucleotide-free form. rho GDI did not stimulate the GTPase activity of rhoB p20 and by itself showed neither GTP gamma S-binding nor GTPase activity. rho GDI was equally active for rhoA p21 and rhoB p20 but was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p25A, and smg p21. rho GDI activity was detected in the cytosol fraction of various rat tissues. These results indicate that, in mammalian tissues, there is a novel type of regulatory protein specific for the rho proteins that interacts with the GDP-bound form of the rho proteins and thereby regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. Since there is a GTPase-activating protein for the rho proteins stimulating the GTPase activity of the rho proteins in mammalian tissues, the rho proteins appear to be regulated at least by GTPase-activating protein and GDI in a dual manner. 相似文献
12.
Alberto Gandarillas Jaime Renart Miguel Quintanilla 《Molecular and cellular biochemistry》1992,112(1):29-33
The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze GP4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s. 相似文献
13.
The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites. 相似文献
14.
T Sasaki A Kikuchi S Araki Y Hata M Isomura S Kuroda Y Takai 《The Journal of biological chemistry》1990,265(4):2333-2337
A novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5'-(3-O-thio)triphosphate (GTPgamma S), from smg p25A. smg p25A GDI also inhibited the binding of GTPgamma S to the GDP-bound form of smg p25A but not of that to the guanine nucleotide-free form. GDI did not stimulate the GTPase activity of smg p25A and by itself showed neither GTPgammaS-binding nor GTPase activity. GDI was inactive for other ras p21/ras p21-like GTP-binding proteins including c-Ha-ras p21, rhoB p20, and smg p21. The Mr value of GDI was estimated to be about 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 65,000 from the S value (4.5 S), and about 82,000 by gel filtration. The isoelectric point of GDI was about pH 5.6. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein. 相似文献
15.
Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli 总被引:6,自引:0,他引:6
M Frech I Schlichting A Wittinghofer P Chardin 《The Journal of biological chemistry》1990,265(11):6353-6359
The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions. 相似文献
16.
We have investigated the kinetics of the binding of guanine nucleotides to bovine brain rhoB p20, a ras p21-like GTP-binding protein with GTPase activity. The initial velocities of the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to GDP-bound rhoB p20 and the dissociation of GDP from this protein were markedly increased by decreasing Mg2+ concentrations. The initial velocity of the binding of GTP gamma S to GDP-free rhoB p20 was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from rhoB p20 limits the binding of GTP to this protein, and suggest that there is a factor stimulating the dissociation of GDP from rhoB p20 and thereby stimulating the binding of GTP to this protein in mammalian tissues. Consistently, the factor stimulating the dissociation of GDP, but not of GTP gamma S, from rhoB p20 was detected in bovine brain cytosol. 相似文献
17.
Ras p21 proteins with high or low GTPase activity can efficiently transform NIH/3T3 cells 总被引:21,自引:0,他引:21
We sought to determine whether decreased in vitro GTPase activity is uniformly associated with ras p21 mutants possessing efficient transforming properties. Normal H-ras p21-[Gly12-Ala59] as well as an H-ras p21-[Gly12-Thr59] mutant exhibited in vitro GTPase activities at least fivefold higher than either H-ras p21-[Lys12-Ala59] or H-ras p21-[Arg12-Thr59] mutants. Microinjection of as much as 6 X 10(6) molecules/cell of bacterially expressed normal H-ras p21 induced no detectable alterations of NIH/3T3 cells. In contrast, inoculation of 4-5 X 10(5) molecules/cell of each p21 mutant induced morphologic alterations and stimulated DNA synthesis. Moreover, the transforming activity of each mutant expressed in a eukaryotic vector was similar and at least 100-fold greater than that of the normal H-ras gene. These findings establish that activation of efficient transforming properties by ras p21 proteins can occur by mechanisms not involving reduced in vitro GTPase activity. 相似文献
18.
Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody. 总被引:1,自引:1,他引:0
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The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 X 10(-8)M for GTP and 1.0 X 10(-8)M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function. 相似文献
19.
M Hiroyoshi K Kaibuchi S Kawamura Y Hata Y Takai 《The Journal of biological chemistry》1991,266(5):2962-2969
Limited proteolysis with trypsin of smg p21B, a ras p21-like small GTP-binding protein having the same putative effector domain as ras p21s, produced the N-terminal fragment and the C-terminal tail of Lys-Lys-Ser-Ser-geranylgeranyl-Cys methyl ester. The Mr values of the intact smg p21B, the N-terminal fragment, and the C-terminal tail were estimated to be about 22,000, 20,500, and less than 1,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the GDP- and GTP-bound forms of the intact smg p21B bound to various membranes and phosphatidylserine-linked Affi-Gel. However, both the GDP- and GTP-bound forms of the N-terminal fragment failed to bind to membranes and phosphatidylserine-linked Affi-Gel. In contrast, the C-terminal tail bound to membranes and phosphatidylserine-linked Affi-Gel. The N-terminal fragment contained a GDP/GTP-binding and GTPase domain and exhibited these two activities, but the C-terminal tail did not show any such activity. A GTPase-activating protein for smg p21 stimulated the GTPase activity of both the intact smg p21B and the N-terminal fragment. In contrast, a GDP/GTP exchange protein for smg p21, named GDP dissociation stimulator, stimulated the GDP/GTP exchange reaction of the intact smg p21B but not that of the N-terminal fragment. These results indicate 1) that smg p21B is composed of at least two functionally different domains, the N-terminal GDP/GTP-binding and GTPase domain and the C-terminal membrane-binding domain, 2) that smg p21B binds to membranes through its C-terminal hydrophobic and basic domain, and 3) that this C-terminal domain is also essential for the smg p21 GDP dissociation stimulator action but not for the smg p21 GTPase-activating protein action. 相似文献
20.
Molecular dynamics simulations of Gly-12-->Val mutant of p21(ras): dynamic inhibition mechanism.
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The mutant p21(ras) protein is a G protein produced by the point-mutated H-ras gene, and this mutant protein has been shown to cause carcinogenesis due to a reduction in its GTPase activity. However, the mechanism underlying this strange phenomenon has still not been elucidated. In our previous study, we have clarified the mechanism of the GTP-->GDP hydrolysis reaction in the wild-type p21(ras) at the atomic level and concluded that GTPase-activating protein plays a significant role in the supply of H2O molecules for the hydrolysis. The structure of the active site in the mutant is the same as that in the wild type. However, by performing molecular dynamic calculations, we found that the structure of the active site of the enzyme substrate complex in the oncogenic mutant p21(ras) continuously changes, and these continuous changes in the active site would make it difficult for the GTP-->GDP hydrolysis reaction to occur in the mutant. These findings can explain the fact that the GTPase activity in the mutant was only 15% of that in the wild type and the fact that GTPase-activating protein has no reaction-activating effect in the mutant. This is a dynamic inhibition mechanism of a vital reaction that can be explained by considering the molecular dynamics. 相似文献