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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions. A burst of GTP hydrolysis, with a kapp value greater than 30 s-1 (20 degrees C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid. The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid. E. coli tRNAPhe, but not N-acetylphenylalanyl-tRNAPhe (N-AcPhe-tRNAPhe), stimulates the GTPase when bound in the P site. If the A site of poly(U)-programmed ribosomes, carrying tRNAPhe in the P site, is occupied by N-AcPhe-tRNAPhe, the burst of Pi discharge is replaced by a slow GTP hydrolysis. Since, under these conditions, N-AcPhe-tRNAPhe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.
J Mol Biol 1986 Jun 20
PMID:Mechanism of ribosomal translocation. Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis. 353 10

Several guanine nucleotide analogs, in one series of which a hydrogen on the 2-amino group is replaced with the p-n-butylphenyl group (BuPGNP derivatives), were used to probe the GTP binding domain of bovine transducin. The order of apparent binding affinities in a series of nucleoside 5'-triphosphates was GTP gamma S greater than GTP approximately BuPGTP greater than dGTP approximately ITP much greater than ATP, values which were 30-100 times higher than affinities of the corresponding 5'-diphosphates. A derivative bearing a 6-aminohexylamino group on the gamma-phosphate, BuPGTP X C6, had a 60-fold lower affinity compared to BuPGTP. In contrast, the p-n-butylphenyl substituent on the 2-amino group had little effect on the binding affinity relative to GTP. Substitutions at the 2-amino group had little effect on either the hydrolysis of the derivatives by the GTPase activity associated with the alpha-subunit of transducin or the activation of cGMP phosphodesterase. The results indicate that the GTP binding domain of transducin is similar in tertiary structure to the corresponding domain of EF-Tu. The 5'-phosphates of GTP are oriented in the binding site of transducin so that the bulky C6 group of BuPGTP X C6 dramatically interferes with binding. The 2-amino group on the guanine ring is probably located at the periphery of the binding site, with the p-n-butylphenyl substituent of BuPGTP facing outward and only weakly interacting with the protein. BuPGTP should be an excellent parent compound for development of novel probes of G-protein interactions with other cellular proteins involved in receptor signal transduction.
Mol Pharmacol 1986 Dec
PMID:Ability of guanine nucleotide derivatives to bind and activate bovine transducin. 353 83

We expressed six forms of p21-ras polypeptides in Escherichia coli with differing transformation potentials resulting from amino acid substitutions at position 12. The ability of the encoded p21's to autophosphorylate, bind guanine nucleotides, and hydrolyze GTP was assessed. All versions of p21 bound GTP equivalently; the kinase activity, while dependent upon residue 12, did not correlate with the transforming potential of the polypeptide. All transforming versions exhibited an impaired GTPase activity, while a novel nontransforming derivative [p21(pro-12)] possessed an enhanced GTPase activity. These results provide strong support for the proposal that an impairment of the cellular p21 GTPase activity can unmask its transforming potential.
Mol Cell Biol 1986 Feb
PMID:Biochemical characterization of polypeptides encoded by mutated human Ha-ras1 genes. 353 94

An Ala-to-Thr substitution at position 59 activates the transforming properties of the p21ras protein without impairment of GTPase activity, a biochemical alteration associated with other activating mutations. To investigate the basis for the transforming properties of the Thr-59 mutant, we characterized guanine nucleotide release. This reaction exhibited a slow rate and stringent temperature requirements. To further dissect the release reaction, we used monoclonal antibodies directed against different epitopes of the p21 molecule. One monoclonal specifically interfered with nucleotide release, while others which recognized different regions of the molecule blocked nucleotide binding. Mutants with the Thr-59 substitution exhibited a three- to ninefold-higher rate of GDP and GTP release than normal p21 or mutants with other activating lesions. This alteration in the Thr-59 mutant would have the effect of increasing its rate of nucleotide exchange. In an intracellular environment with a high GTP/GDP ratio, this would favor the association of GTP with the Thr-59 mutant. Consistent with knowledge of known G-regulatory proteins, these findings support a model in which the p21-GTP complex is the biologically active form of the p21 protein.
Mol Cell Biol 1986 Dec
PMID:Activation of ras p21 transforming properties associated with an increase in the release rate of bound guanine nucleotide. 354 Jun 8

We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.
Mol Cell Biol 1987 Jan
PMID:Biochemical and biological properties of the human N-ras p21 protein. 355 Apr 23

The high prevalence of ras oncogenes in human tumors has given increasing impetus to efforts aimed at elucidating the structure and function of their p21 products. To identify functionally important domains of the p21 protein, antibodies were generated against synthetic peptides corresponding to various regions of the protein. Antibodies directed against a synthetic peptide fragment corresponding to amino acid residues 161 to 176 in the carboxy-terminal region of the H-ras-encoded p21 molecule specifically recognized H-ras-encoded p21 proteins. This antibody was also shown to strikingly and specifically inhibit the guanine nucleotide-binding function of the p21 protein. The inability of p21 protein to bind guanine nucleotides was associated with a lack of autophosphorylation or GTPase activities. These studies suggest that a region toward its carboxy terminus is directly or indirectly involved in the guanine nucleotide-binding function of the p21 molecule.
Mol Cell Biol 1985 Nov
PMID:Antibody of predetermined specificity to a carboxy-terminal region of H-ras gene products inhibits their guanine nucleotide-binding function. 391 72

The influence of an acute exposure to ethanol on adenylyl cyclase activity in membrane fractions prepared from human corpus luteum was investigated. Ethanol up to a concentration of 5% (v/v) was without effect on basal luteal adenylyl cyclase activity, but markedly potentiated stimulation of NaF and hCG in a dose-dependent manner. In contrast, ethanol progressively inhibited forskolin stimulation at the same range of ethanol concentrations. Maximal NaF and hCG responsiveness of adenylyl cyclase activity was observed at 5% ethanol and reached values 80% and 100% higher than controls without ethanol, respectively. However, at the same ethanol concentration, forskolin-stimulated enzymatic activity was reduced by 40% relative to controls. Equilibrium binding studies involving [125I]hCG interaction with luteal membranes in the presence of the concentration of ethanol showing maximal hCG responsiveness indicated that ethanol slightly affected (15% increase) the hCG binding compared to controls, without any appreciable change on the Kd for the hormone. This minor effect of ethanol on gonadotropin binding sites contrasted greatly with the extent at which ethanol maximally potentiated the gonadotropin-stimulated adenylyl cyclase. GTP was found to be less effective than GMP-P(NH)P in sustaining ethanol potentiation, suggesting that ethanol is unlikely to act by inhibiting GTPase activity. These data indicate that the acute effects of ethanol inhibit forskolin-stimulated adenylyl cyclase at concentrations potentiating stimulatory effects of NaF and of hCG, and that the synergistic interaction of ethanol and gonadotropin stimulation of adenylyl cyclase is, at least in part, due to an increase in the functional coupling of the occupied hCG-receptor complex with the components of the enzyme system.
Mol Cell Endocrinol 1985 May
PMID:Opposite effects of ethanol on the activation of adenylyl cyclase in human corpus luteum membranes. 404 41

During protein synthesis the interaction with ribosomes of elongation factors Tu (EF-Tu), G (EF-G) and initiation factor 2 (IF-2) is associated with the hydrolysis of GTP which is directly related to the functions of the factors. In this article we review systematically the properties of these GTPase activities in the presence and absence of protein synthesis, and by examining the characteristics of the different minimal systems for the expression of these activities we point to the role of the various effectors and to the enzymological aspects of the systems. For EF-Tu, it has been possible to eliminate any requirement for macromolecular effectors, showing that the factor itself is a GTPase. For EF-G, the presence of at least the 50S ribosomal subunit has remained a requirement, whereas IF-2 needs both the 50S and 30S subunits to exhibit GTPase activity. Between the GTPase activities of the three factors there are some striking similarities, but important differences prevail as a consequence of the specificity of the different functions. This can also be seen by examining the respective ribosomal regions implicated in these reactions. When coupled with protein synthesis, the three GTPase activities reveal characteristics differing from those observed in partial systems.
Mol Cell Biochem 1981 Mar 27
PMID:Properties and regulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2. 611 39

The activity of enzymes exhibiting GTPase activity was determined in membrane preparations of hamster adipocytes. Two GTPases with apparent Km values of about 0.2 microM GTP (low Km GTPase) and 180 microM GTP (high Km GTPase) were found. The effects of various agents that stimulate or inhibit adipocyte adenylate cyclase were investigated with these two forms of GTPase. None of the stimulatory or inhibitory agonists studied affected the activity of the high Km GTPase(s). However, factors inhibiting adenylate cyclase, such as prostaglandin E1, nicotinic acid, 3-carboxy-5-methylpyrazole, and N6-phenylisopropyladenosine, stimulated the low Km GTPase by 50--100% without an apparent lag phase. The activity of the stimulated GTPase was half-maximal at about 0.2 microM GTP. NaCl (up to 100 mM) had no effect on the basal activity of this enzyme but amplified the stimulation induced by adenylate cyclase inhibitory agents. There was a good correlation between inhibition of adenylate cyclase and stimulation of the low Km GTPase, both with regard to the concentration required for half-maximal effects on these two enzymes and with regard to the potency order of various prostaglandins studied. In contrast to factors inhibiting adenylate cyclase, the stimulatory hormones, isoproterenol and ACTH, had only small effects on the low Km GTPase activity; potassium fluoride was completely ineffective. The data suggest that an increased GTP hydrolysis by an activated GTPase is an essential mechanism of hormone-induced adenylate cyclase inhibition.
Mol Pharmacol 1982 Mar
PMID:Stimulation of a low Km GTPase by inhibitors of adipocyte adenylate cyclase. 612 77

The influence of somatostatin was studied on GTPase activity in membranes of cyc- and H21a variants of S49 lymphoma cells, which are functionally defective in the guanine nucleotide site (Ns) mediating hormonal stimulation of the adenylate cyclase. Somatostatin, which inhibits adenylate cyclase in these membranes by a GTP-dependent process, caused a concomitant activation of a high-affinity GTPase (apparent Km approximately equal to 0.2 microM) by 40-50%. The hormone-stimulated GTPase also exhibited an apparent Km value of about 0.2 microM. GTPase stimulation by somatostatin occurred without an apparent lag phase. There was a close correlation between adenylate cyclase inhibition and high-affinity GTPase stimulation induced by somatostatin. Various other peptide hormones studied and isoproterenol had no effect on GTP hydrolysis. Activation of the enzyme by somatostatin was reduced or abolished by pretreatment of the membranes with the SH reagent, N-ethylmaleimide. In membranes of wild-type S49 lymphoma cells, somatostatin caused an increase in GTPase activity similar to that in cyc- and H21a membranes. The data show that cyc- and H21a membranes, which are more or less defective in Ns, contain a hormone-sensitive, high-affinity GTPase and that the activation of this enzyme is closely related to adenylate cyclase inhibition by somatostatin. The data suggest that, similar to Ns, the activity state of the guanine nucleotide site (Ni), which apparently mediates somatostatin-induced inhibition of the adenylate cyclase, is controlled by a high-affinity GTPase.
Mol Pharmacol 1983 Sep
PMID:Somatostatin-induced stimulation of a high-affinity GTPase in membranes of S49 lymphoma cyc- and H21a variants. 613 2


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