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1.
ATP sulfurylase catalyzes and couples the free energies of two reactions: GTP hydrolysis and the synthesis of activated sulfate, or APS. The GTPase active site undergoes changes during its catalytic cycle that are driven by events that occur at the APS-forming active site, which is located in a separate subunit. GTP responds to its changing environment by moving along its reaction path. The response, which may change the affinity or reactivity of GTP, can, in turn, produce alterations at the APS active site that drive APS synthesis. The resulting stepwise progression of the two reactions couples their free energies. The mechanism of ATP sulfurylase involves an enzyme isomerization that precedes and rate limits cleavage of the beta,gamma-bond of GTP. These fluorescence studies demonstrate that the isomerization is controlled by the binding of activators that drive ATP sulfurylase into forms that mimic different stages of the APS reaction. Only certain activators elicit the isomerization, suggesting that the APS reaction must proceed to a specific point in the catalytic cycle before the conformational "switch" that controls GTP hydrolysis is thrown. The isomerization is shown to require occupancy of the gamma-phosphate subsite of the GTP binding pocket. This requirement establishes that the isomerization results in a change in the interaction between the enzyme and the gamma-phosphate of GTP that emerges in the catalytic cycle during the transition from the nonisomerized to the isomerized E.GTP complex. The newly formed contact(s) appears to carry into the bond-breaking transition state, and to be essential for the enhanced affinity and reactivity of the nucleotide. 相似文献
2.
Sun M Andreassi JL Liu S Pinto R Triccas JA Leyh TS 《The Journal of biological chemistry》2005,280(9):7861-7866
The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5'-phosphosulfate (APS), the hydrolysis of GTP, and the 3'-phosphorylation of APS to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E.P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 10(6)-fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy-coupling isomerization. 相似文献
3.
A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J. F., & Webb, M. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565]. We have now shown that the rate of the fluorescence change is accelerated by GTPase-activating protein (GAP) in the same manner as that of the GTP cleavage reaction. In contrast, a faster fluorescence change of smaller amplitude seen in the complex between p21 and the uncleavable 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-O-(beta,gamma-imidotriphosphate) (mGppNHp) is not affected by GAP. The corresponding fluorescent derivative of guanosine 5'-O-(gamma-thiotriphosphate) (mGTP gamma S) shows a very slow fluorescence change after binding to p21, and this rate is also accelerated significantly by GAP. Hydrolysis of GTP gamma S is similarly slow, and it is accelerated by GAP in a similar manner to the fluorescence change. The results are interpreted to indicate that the fluorescence change occurs either at the hydrolysis step or on release of inorganic phosphate or thiophosphate but does not occur in a rate-limiting step preceding hydrolysis. 相似文献
4.
The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site. 相似文献
5.
Anatomy of an energy-coupling mechanism--the interlocking catalytic cycles of the ATP sulfurylase-GTPase system 总被引:1,自引:0,他引:1
ATP sulfurylase, from Escherichia coli K-12, conformationally couples the rates and chemical potentials of the two reactions that it catalyzes, GTP hydrolysis and activated sulfate synthesis. The enzyme is rare among such coupling systems in that it links the potentials of small-molecule chemistries to one another, rather than to vectorial motion. The pre-steady-state stages of the catalytic cycle of ATP sulfurylase were studied using tools capable of distinguishing between enzyme-bound and solution-phase product for each of the four products of the enzyme. The study reveals that the two chemistries are linked at multiple points in the reaction coordinate. Linking begins with an isomerization prior to chemistry that initiates an ordered cleavage of the beta,gamma and alpha,beta bonds of GTP and ATP, respectively; the rates of these three sequential events increase successively, causing them to appear simultaneous. Linking is again seen in the late stages of the catalytic cycle: product release is ordered with P(i) departing prior to either GDP or PP(i). Release rate constants are determined for each product and used to construct a quantitative model of the mechanism that accurately predicts the behavior of this complex system. 相似文献
6.
ATP sulfurylase, from Escherichia coli K-12, catalyzes and couples the Gibbs potentials of two reactions, GTP hydrolysis and activated sulfate (APS, adenosine 5'-phosphosulfate) synthesis. Coupling these potentials requires that the catalytic cycle include reaction stage-dependent conformational changes that gate the activities of the two active sites. These interactions were probed in a mutagenesis study of a highly conserved pyrophosphate-binding motif (SXGXDS), which is located at the APS-forming active site. The motif appears to be unique to the N-type PPi synthetase family, and mutations in it are linked, in other systems, to citrullinemia, an often fatal orphan disease. The conserved sites in the motif were evaluated individually for their ability to activate GTP hydrolysis (which reports interactions among the activator (AMP or Mg2+.PPi), the enzyme, and GTP), to affect the energetic coupling of the two reactions, and to alter the kinetic constants of the adenylyl transfer reaction in the absence of guanine nucleotide. What emerges from this first mutagenic exploration of the PPi motif in any adenylyltransferase is that the residues of the motif participate differently, and in sometimes profoundly important ways, in the different functions of the enzyme. 相似文献
7.
Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome. 总被引:8,自引:4,他引:4
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The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis. 相似文献
8.
An analysis of the role of guanine nucleotide binding proteins in antigen receptor/CD3 antigen coupling to phospholipase C. 总被引:1,自引:0,他引:1
In permeabilized human T lymphocytes, phospholipase C (PLC)-mediated metabolism of polyphosphatidylinositols can be stimulated by triggering the T cell antigen receptor/CD3 antigen complex (Ti/CD3) with the CD3 antibody UCHT1 or by activation of G proteins with the non-hydrolyzable guanine nucleotide analogue, guanosine 5'-O-(3-thiotrisphosphate) (GTP[S]). Ti/CD3 induction of inositol phosphate production demonstrated no dependence on exogenous guanine nucleotides. Furthermore, Ti/CD3 stimulation did not influence the kinetics or dose-response of GTP[S]-induced inositol phosphate production, suggesting that the Ti/CD3 complex does not regulate guanine nucleotide exchange on the G protein pool stimulated by GTP[S]. These data indicate that the Ti/CD3 complex is not G protein-linked to PLC in a manner analogous to the G protein linkage of receptors to adenylate cyclase. However, the inhibitory guanine nucleotide, GDP, antagonizes not only GTP[S]-induced polyphosphatidylinositol hydrolysis but also UCHT1-induced inositol phosphate production. These data infer that a G protein can modulate the coupling of the Ti/CD3 complex to PLC and that there may be some "cross-talk" between Ti/CD3 and G protein PLC coupling mechanisms. 相似文献
9.
Binns DD Helms MK Barylko B Davis CT Jameson DM Albanesi JP Eccleston JF 《Biochemistry》2000,39(24):7188-7196
Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility. 相似文献
10.
Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins 总被引:4,自引:0,他引:4
Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C. 相似文献
11.
Ca2+ inhibits guanine nucleotide-activated phospholipase D in neural-derived NG108-15 cells.
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We have investigated the regulation of phospholipase D (PLD) activity by guanine nucleotides and Ca2+ in cells of the NG108-15 neuroblastoma X glioma line that were permeabilized with digitonin. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) caused a nearly sixfold increase (EC50 = 3 microM) in production of [3H]phosphatidylethanol (specific product of the PLD transphosphatidylation reaction). Other GTP analogues were less effective than GTP gamma S, and guanosine-5'-O-(2-thiodiphosphate) inhibited PLD activation by GTP gamma S. Both basal and GTP gamma S-stimulated PLD activities were potentiated by MgATP and Mg2+. Adenosine-5'-O-(3-thiotriphosphate) and ADP also potentiated the effect of GTP gamma S, but non-phosphorylating analogues of ATP had no such effect. The activation of PLD by GTP gamma S did not require Ca2+ and was independent of free Ca2+ ions up to a concentration of 100 nM (resting intracellular concentration). Higher Ca2+ concentrations (greater than or equal to 1 microM) completely inhibited PLD activation by GTP gamma S. It is concluded that elevated intracellular Ca2+ concentrations may negatively modulate PLD activation by a guanine nucleotide-binding protein, thus affecting receptor-PLD coupling in neural-derived cells. 相似文献
12.
Differential mechanisms of inositol phosphate generation at the receptors for bombesin and platelet-derived growth factor. 总被引:3,自引:0,他引:3
We investigated the mechanism(s) whereby activation of a growth-factor receptor typically endowed with tyrosine kinase activity, such as the platelet-derived growth factor (PDGF) receptor, triggers phosphoinositide hydrolysis. In Swiss 3T3 cells permeabilized with streptolysin O, an analogue of GTP, guanosine 5'-[gamma-thio]triphosphate, was found to potentiate the coupling of the bombesin receptor to phospholipase C. In contrast, the activation of the enzyme by PDGF occurred in a GTP-independent manner. Moreover, the inactive analogue of GTP, guanosine 5'-[beta-thio]diphosphate, significantly inhibited the bombesin-induced InsP3 generation, whereas it did not decrease the same effect when stimulated by PDGF. 相似文献
13.
G Proteins Coupled to Phospholipase C: Molecular Targets of Long-Term Ethanol Exposure 总被引:1,自引:0,他引:1
Per Simonsson F. David Rodriguez Niklas Loman Christer Alling 《Journal of neurochemistry》1991,56(6):2018-2026
Long-term ethanol exposure is known to inhibit bradykinin-stimulated phosphoinositide hydrolysis in cultures of neuroblastoma x glioma 108-15 cells. In the present study, [3H]bradykinin binding, GTP-binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5'-O-(3-thiotriphosphate) [GTP(S)]- and, to a lesser extent, NaF/AlCl3-stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol-containing medium. The results indicate that long-term ethanol exposure exerts its effects on receptor-stimulated phosphoinositide hydrolysis primarily at the level of the GTP-binding protein. 相似文献
14.
T F Martin S M Bajjalieh D O Lucas J A Kowalchyk 《The Journal of biological chemistry》1986,261(22):10141-10149
Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP). 相似文献
15.
Guanine nucleotides stimulate soluble phosphoinositide-specific phospholipase C in the absence of membranes 总被引:12,自引:0,他引:12
The effect of guanine nucleotides on platelet and calf brain cytosolic phospholipase C was examined in the absence of membranes or detergents in an assay using labeled lipid vesicles. Guanine nucleotides stimulate hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PtdIns-4,5-P2) catalyzed both by enzyme from human platelets and by partially purified enzyme from calf brain. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was the most potent guanine nucleotide with a half-maximal stimulation at 1-10 microM, followed by guanosine 5'-(beta, gamma-imido)triphosphate greater than GTP greater than GDP = guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(2-thiodiphosphate) was able to reverse the GTP gamma S-mediated stimulation. NaF also stimulated phospholipase C activity, further implying a role for a guanine nucleotide-binding protein. In the presence of GTP gamma S, the enzyme cleaved PtdIns-4,5-P2 at higher pH values, and the need for calcium ions was reduced 100-fold. The stimulation of PtdIns-4,5-P2 hydrolysis by GTP gamma S ranged from 2 to 25-fold under various conditions, whereas hydrolysis of [3H]phosphatidylinositol was only slightly affected by guanine nucleotides. We propose that a soluble guanine nucleotide-dependent protein activates phospholipase C to hydrolyze its initial substrate in the sequence of phosphoinositide-derived messenger generation. 相似文献
16.
GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism. The activation of the enzyme, purified from Escherichia coli, is titratable with GTP. The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP. The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis. The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins. This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase. 相似文献
17.
We have studied the effect of guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), a non-hydrolyzable analogue of GTP, on agonist and antagonist binding to bovine hippocampal 5-hydroxytryptamine (5-HT)(1A) receptor in native membranes. Our results show that the specific binding of the agonist is inhibited with increasing concentrations of GTP-gamma-S along with a reduction in binding affinity. In sharp contrast to this, antagonist binding to 5-HT(1A) receptor shows no significant reduction and remains invariant over a large range of GTP-gamma-S concentrations. The binding affinity of the antagonist also remains unaltered. This shows that the agonist and the antagonist differentially discriminate G-protein coupling of 5-HT(1A) receptors from bovine hippocampus. 相似文献
18.
The interplay between activated G proteins and intracellular calcium ([Ca(2+)](i)) in the regulation of secretion was studied in the macrophage, coupling membrane capacitance with calcium-sensitive microfluorimetry. Intracellular elevation of either the nonhydrolyzable analogue of GTP, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), or [Ca(2+)](i) enhanced the amplitude and shortened the time course of stimulus-induced secretion in a dose-dependent manner. Both the ionophore- and the stimulus-induced secretory response were abolished in the presence of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The K(d) of Ca(2+)-driven secretion was independent of GTP gamma S concentration, whereas the K(d) of the GTP gamma S-driven response decreased from 63 to 31 microM in the presence of saturating concentrations of [Ca(2+)](i). The time course of stimulus-induced secretion was dependent upon the concentration of [Ca(2+)](i). The time course of GTP gamma S-driven secretion was concentration-independent at high levels of [Ca(2+)](i), suggesting that a calcium-dependent translocation/binding step was rate-limiting. Our data strongly support a model in which [Ca(2+)](i) and activated G proteins act independently of one another in the sequential regulation of macrophage secretion. [Ca(2+)](i) appears to play a role in the recruitment and priming of vesicles from reserve intracellular pools at a step that is upstream of G protein activation. While activated, G proteins appear to play a key role in fusion of docked vesicles. Thus, secretion can result either from activating more G proteins or from elevating [Ca(2+)](i) at basal levels of G protein activation. 相似文献
19.
Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly. 相似文献
20.
Rabbit brain cortical membranes, which have been extracted with 2 M KCl, hydrolyze exogenously added [3H]phosphatidylinositol [( 3H]PI) in a guanine nucleotide- and carbachol-dependent manner. Both oxotremorine-M and carbachol are full agonists with EC50 values of 8 and 73 microM, respectively. Pirenzepine and atropine inhibit carbachol-stimulated [3H]PI hydrolysis. The hydrolysis-resistant guanine nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is the most potent in supporting carbachol-stimulated hydrolysis of PI. There is no effect of carbachol in the absence of guanine nucleotides or in the presence of 100 microM adenosine 5'-O-(3-thiotriphosphate), adenosine-5'-(beta, gamma-imido)triphosphate, or sodium pyrophosphate. Guanylyl-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] in the presence of carbachol also stimulates PI hydrolysis although much less than that seen with GTP gamma S. GDP and Gpp(NH)p are potent antagonists of the GTP gamma S-dependent carbachol response. Optimal stimulation by carbachol and GTP gamma S was observed at 0.3-1 microM free Ca2+ and 6 mM MgCl2. Limited trypsinization resulted in loss of receptor-regulated PI breakdown and a slight decrease in basal activity. These results demonstrate that phospholipase C hydrolysis of exogenous PI by rabbit cortical membranes may be stimulated by carbachol in a guanine nucleotide-dependent manner. 相似文献