The modulation of Ca-ATPase activity and protein-lipid interactions in the sarcoplasmic reticulum by ATP

The time-course of ATP hydrolysis by Ca-ATPase of purified sarcoplasmic reticulum is biphasic with an initial rate over 1 to 2 min exceeding the subsequent rate. Hydrolysis of GTP and p-nitrophenylphosphate (pNPP) occurs at a slower but constant rate. Arrhenius plots of GTP, p-nitrophenylphosphate and initial rates of ATP hydrolysis all exhibit a discontinuity at about 20-24 degrees C; no breaks are observed in plots of the slower phase of ATP hydrolysis. The effect of substrate hydrolysis on the disposition of the enzyme in the membrane was examined by monitoring the quenching of tryptophan fluorescence by pyrene present in the hydrophobic domain of the membrane. The presence of ATP, but not GTP, prevents a temperature-dependent decrease in fluorescence quenching suggesting that ATP binding causes a change in the protein domain in contact with the membrane lipids.     

Tubulin polymerization with ATP is mediated through the exchangeable GTP site

Glycerol-induced tubulin polymerization supported by non-guanine nucleotides was examined. The electrophoretically homogeneous tubulin was devoid of nucleoside diphosphate kinase activity and 95% saturated with exchangeable GDP and nonexchangeable GTP. All purine ribonucleoside 5'-triphosphates were active but no polymerization occurred with CTP or UTP. All polymerization reactions, as a function of nucleotide concentration, were similar: above a minimum (threshold) concentration, as the amount of nucleotide increased the reaction became progressively more rapid and extensive with a progressively shorter nucleation period. Threshold concentrations of ATP, XTP, ITP and GTP were 0.6 mM, 0.3 mM, 30 microM and 7 microM, respectively. Most ribose- and polyphosphate-modified ATP analogs also supported polymerization at high concentrations, but the activity of these analogs relative to ATP was very similar to the activity of cognate GTP analogs relative to GTP. Polymerization with ATP was associated with an ATPase reaction. ATP hydrolysis was potently inhibited by GDP and GTP and altered by antimitotic drugs in parallel with the effects of these agents on GTP hydrolysis. Substantial amounts of [8-14C]GDP bound in the exchangeable site of tubulin were displaced during polymerization with GTP or ATP, but much higher concentrations of ATP were required for equivalent displacement of the tubulin-bound GDP. Polymerization with GTP or ATP was inhibited in a qualitatively similar manner by GDP, with increasing concentrations of GDP causing a progressive prolongation of the nucleation period and reduction in reaction rate and extent. However, complete inhibition of polymerization required that GDP:GTP much greater than 1, but that GDP:ATP much less than 1. Inhibition appeared to be primarily competitive, since with higher triphosphate concentrations higher GDP concentrations were required for comparable inhibition. We conclude that ATP effects on tubulin polymerization are mediated through a feeble interaction at the exchangeable GTP site.     

The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates

ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 1. Three-strand exchange reaction.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.     

In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.     

Molecular basis for differential nucleotide binding of the nucleotide-binding domain of ABC-transporter CvaB

The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC-transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP and, indeed, highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide-binding domain predicted that the aromatic stacking region of CvaB (Y501DSQ loop) had a role in the differential binding of nucleotides, and Ser503 and Gln504 provided potential hydrogen bonds to GTP but not to ATP. Site-directed mutagenesis of the Y501DSQ loop, mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature effect assays on nucleotide binding and hydrolysis further indicated that the double mutant protein had largely eliminated the difference for substrates ATP and GTP, and behaved more similarly to the NBD of typical ABC-transporter HlyB. Therefore, we conclude that Ser503 and Gln504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference.     

Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells.

Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with Km and Vmax values similar to those for ATP (0.9-1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gamma S and AMPPNP, and weakly by AMPCPP and ADP-beta S, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-32P]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed to be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.     

Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate)

Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.     

Nucleotide specificity of cardiac sarcoplasmic reticulum. GTP-induced calcium accumulation and GTPase activity

We previously demonstrated that the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium and does not support the translocation of calcium and oxalate into the vesicular space. In response to GTP, however, calcium is accumulated into a compartment which is sensitive to pH and ionophore. In the present paper, we further explored the relationship between GTP hydrolysis and GTP-induced calcium accumulation. Both ATP- and GTP-induced calcium accumulation were prevented by the sulfhydryl reagent, N-ethylmaleimide (NEM; I50 = 0.2 mM). In contrast, the sensitivity of NTP hydrolysis to NEM differed markedly; GTPase activity was not affected by NEM, whereas ATPase activity was markedly inhibited. Conversely, although the GTPase was noncompetitively inhibited by the ATP analogue, adenylyl imidodiphosphate (Ki = 8 microM), and was competitively inhibited by the GTP analogue, guanylyl imidodiphosphate (Ki = 60 microM), GTP-induced calcium accumulation was not affected by the NTP analogues at any concentration. Therefore, the GTP-dependent accumulation of calcium into the pH- and ionophore-sensitive compartment of cardiac SR may not require GTP hydrolysis but may be dependent on GTP binding. The previously reported noncompetitive inhibition of the GTPase by ATP was also observed when the calcium-dependent hydrolysis of ATP was prevented by NEM (Ki = 1.2 microM). Along with the noncompetitive inhibition of the GTPase by adenylyl imidodiphosphate, the inhibition of the GTP by ATP in the presence of NEM suggests that ATP binding may be involved in the observed inhibition. The Ki for the noncompetitive inhibition of GTPase activity is compatible with ATP binding to the high affinity catalytic site of the ATPase. Thus, although GTP-induced calcium accumulation differs somewhat from ATP-dependent calcium translocation, the similarities between the two processes (i.e. similar time courses and sensitivity to pH, ionophore, and sulfhydryl modification) suggest that they may be related in some manner.     

Identification and characterization of a hitherto unknown nucleotide-binding domain and an intricate interdomain regulation in HflX-a ribosome binding GTPase

A role for HflX in 50S-biogenesis was suggested based on its similarity to other GTPases involved in this process. It possesses a G-domain, flanked by uncharacterized N- and C-terminal domains. Intriguingly, Escherichia coli HflX was shown to hydrolyze both GTP and adenosine triphosphate (ATP), and it was unclear whether G-domain alone would explain ATP hydrolysis too. Here, based on structural bioinformatics analysis, we suspected the possible existence of an additional nucleotide-binding domain (ND1) at the N-terminus. Biochemical studies affirm that this domain is capable of hydrolyzing ATP and GTP. Surprisingly, not only ND1 but also the G-domain (ND2) can hydrolyze GTP and ATP too. Further; we recognize that ND1 and ND2 influence each other’s hydrolysis activities via two salt bridges, i.e. E29-R257 and Q28-N207. It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements. Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2. Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.     

ATP-stimulated hydrolysis of GTP by RecA protein: kinetic consequences of cooperative RecA protein-ATP interactions

The cooperativity of the single-stranded DNA dependent nucleoside triphosphatase activity of the recA protein was investigated by examining the influence of a good substrate (ATP) on the hydrolysis of a poor substrate (GTP). At pH 7.5 and 37 degrees C, both ATP and GTP are hydrolyzed with a turnover number of 17.5 min-1. The S0.5 for GTP (750 microM), however, is nearly 20-fold higher than the S0.5 for ATP (45 microM). Low concentrations of ATP activate the GTPase activity of the recA protein by lowering the S0.5 for GTP; in the presence of 50 microM ATP, the S0.5 for GTP is reduced from 750 microM to 200 microM. Concentrations of ATP greater than 50 microM result in competitive inhibition of the ATP-activated GTPase activity. Although GTP is a substrate for hydrolysis, it will not substitute for ATP as a high-energy cofactor in the standard recA protein promoted three-strand exchange reaction. To account for these results, a minimal kinetic model is presented in which ATP binding induces specific conformational changes in the recA protein that do not occur with GTP binding.     

GTP stimulates pregnenolone generation in isolated rat adrenal mitochondria

Pregnenolone synthesis from cholesterol by adrenal mitochondria isolated from ether-stressed rats exhibits a biphasic time course: upon the addition of a reducing substrate (e.g. malate), a rapid phase of pregnenolone formation occurs during the first 5 min, which has been interpreted as the metabolism of a steroidogenic pool of cholesterol, probably in the inner membrane. A slower rate follows, which is interpreted as translocation of cholesterol into the steroidogenic pool. While a 30-min preincubation of mitochondria with cholesterol alone did not affect the extent of the rapid phase, preincubation with GTP plus cholesterol extended the first phase, resulting in an up to 2-fold increase in pregnenolone synthesis by 20-30 min. The apparent Km for GTP was 0.1-0.4 mM, and stimulation was maximal with preincubation times of 10-30 min, depending upon incubation conditions. Exogenous cholesterol was not required to observe a stimulatory effect, indicating that GTP reorganizes the endogenous mitochondrial cholesterol pools. Nevertheless, stimulation was greater when exogenous cholesterol was provided, consistent with enhanced utilization of both endogenous and exogenous cholesterol. Stimulation by GTP was also seen in mitochondria isolated from cycloheximide-injected/ether-stressed rats, although the activity in these preparations was always lower than that in mitochondria from ether-stressed rats. The stimulation was specific for GTP, since many other nucleotides (e.g. ATP, GDP, and ITP) and GTP analogues (guanosine 5'-O-(3-thiotriphosphate and guanosine 5'-(beta,gamma-imino)triphosphate) had no effect. The GTP-activated state was reversible: after GTP hydrolysis by a mitochondrial GTPase, pregnenolone synthesis returned to the basal level. Sonic disruption of mitochondria abolished the stimulatory effect of GTP. These results suggest that GTP enhances pregnenolone synthesis by promoting the movement of cholesterol to the steroidogenic pool, consistent with a recently proposed general role for GTP in some vectorial transport processes (Bourne, H. R. (1988) Cell 53, 669-671).     

In Vitro Generation from the trans-Golgi Network of Coatomer-Coated Vesicles Containing Sialylated Vesicular Stomatitis Virus-G Protein

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin–Darby canine kidney (MDCK) cells in which the ³⁵S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20°C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37°C and does not occur below 20°C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors—one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPγS or GMP–PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20°C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.     

The energetics of ion distribution: the origin of the resting electric potential of cells

The relation between the energies of ion movement and ATP hydrolysis is unknown in tissues with widely varying electric potentials. Consequently, we measured the concentration of the nine major inorganic ions in the extra- and intracellular phases in heart, liver, and red cells with resting electrical potentials, E(N), of -86, -28, and -6 mV, respectively, under six different physiological conditions. We calculated the Nernst electric potential and the energy of ion movement between the phases. We found that the energy of ATP hydrolysis was essentially constant, between -54 and -58 kJ/mol, in all tissues and conditions. In contrast, as E(N) decreased, the energies of the Na+ and K+ gradients decreased, with slopes approximating their valence. The difference between the energies of Na+ and K+ gradients remained constant at 17 kJ/mol, which is approximately one third of the energy of ATP hydrolysis, demonstrating near-equilibrium of the Na+/K+ ATPase in all tissues under all conditions. All cations, except K+, were pumped out of cells and all anions, except Cl- in liver and red cell, were pumped into cells. We conclude that the energy of ATP was expressed in Na+/K+ ATPase and its linked inorganic ion transporters to create a Gibbs-Donnan near-equilibrium system, an inherent part of which was the electric potential.     

The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger.     

Novel activity of eukaryotic translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with GTP

Ribosomes must dissociate into subunits in order to begin protein biosynthesis. The enzymes that catalyze this fundamental process in eukaryotes remained unknown. Here, we demonstrate that eukaryotic translocase, eEF2, which catalyzes peptide elongation in the presence of GTP, dissociates yeast 80S ribosomes into subunits in the presence of ATP but not GTP or other nucleoside triphosphates. Dissociation was detected by light scattering or ultracentrifugation after the split subunits were stabilized. ATP was hydrolyzed during the eEF2-dependent dissociation, while a non-hydrolyzable analog of ATP was inactive in ribosome splitting by eEF2. GTP inhibited not only ATP hydrolysis but also dissociation. Sordarin, a fungal eEF2 inhibitor, averted the splitting but stimulated ATP hydrolysis. Another elongation inhibitor, cycloheximide, also prevented eEF2/ATP-dependent splitting, while the inhibitory effect of fusidic acid on the splitting was nominal. Upon dissociation of the 80S ribosome, eEF2 was found on the subunits. We propose that the dissociation activity of eEF2/ATP plays a role in mobilizing 80S ribosomes for protein synthesis during the shift up of physiological conditions.     

Nucleotide triphosphatase activity of the N-terminal nucleotide-binding domains of the multidrug resistance proteins P-glycoprotein and MRP1

The multidrug resistance proteins P-glycoprotein (Pgp) and MRP1 are drug-efflux pumps. In this study, we compared the nucleotide triphosphatase activities of the isolated N-terminal nucleotide binding domains (NBD1) of Pgp and MRP1, and explored the potential role of the phosphorylation target domain of Pgp on the regulation of Pgp NBD1 ATPase activity. We found that: (1) the NBD1s of Pgp and MRP1 have ATPase and GTPase activities, (2) the K(m)s of Pgp NBD1 for ATP and GTP hydrolysis are identical, while the K(m) of MRP1 NBD1 for ATP is lower than that for GTP, and (3) phosphorylation of MLD by PKA or PKC produces a marginal increase of V(max) for ATP hydrolysis, without affecting the affinity for ATP. These results show efficient GTP hydrolysis by the NBD1s of Pgp and MRP1, and a minor role of phosphorylation in the control of Pgp NBD1 ATPase activity.     

Mutagenesis suggests several roles of Snu114p in pre-mRNA splicing

Snu114p, a yeast U5 small nuclear ribonucleoprotein (snRNP) homologous to the ribosomal GTPase EF-2, was recently found to play a part in the dissociation of U4 small nuclear RNA (snRNA) from U6 snRNA. Here, we show that purified Snu114p binds GTP specifically. To test the possibility that binding and hydrolysis of GTP by Snu114p are required to stimulate the unwinding of U4 from U6, we produced several mutations of Snu114p. Residues whose mutations led to lethal phenotypes were all clustered in the P loop and in the guanine-ring binding sequence (NKXD) of the G domain, which in elongation factor-G is required for the binding and hydrolysis of GTP. An arginine residue in domain II, which in EF-G forms a salt bridge with a residue of the G domain, when mutated in Snu114p (R487E), led to a temperature-sensitive phenotype. The substitution D271N in the NKXD sequence is predicted to bind XTP instead of GTP. Spliceosomes containing this mutant, isolated by affinity chromatography after heat treatment, retained U4 snRNA paired with the U6 snRNA. U4 snRNA was released efficiently only when these arrested spliceosomes were reactivated by lowering the temperature in the presence of a mixture of ATP and XTP. Because non-hydrolyzable XTP analogues did not consent the release of U4, we conclude that the release requires hydrolysis of XTP. This suggests that Snu114p needs GTP to influence, directly or indirectly, the unwinding of U4 from U6. An additional role for Snu114p is also demonstrated: after growth of the D271N and R487E strains at high temperatures, we observed decreased levels of the U5 and the U4/U6.U5 snRNPs. This indicates that, before splicing, Snu114p plays a part in the assembly of both particles.     

Effects of organic acids on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis

We have examined the effects of a number of organic anions, which stabilize tubulin, on tubulin polymerization, associated GTP hydrolysis, and polymer morphology. While microtubule-associated proteins, as well as glycerol, induced formation of typical microtubules in a reaction coupled to GTP hydrolysis at an initial 1:1 stoichiometry, the organic anions had varying effects. Only 2-(N-morpholino)ethanesulfonate induced formation of structures with the morphology of microtubules. With glutamate, fructose 1,6-bisphosphate, piperazine-N-N'-bis(2-ethanesulfonate), glutarate, and glucose 1-phosphate, the predominant structures formed were sheets of parallel protofilaments rather than microtubules. Creatine phosphate induced the formation of clusters of rings. GTP hydrolysis was closely coupled to polymerization only with glutamate. With creatine phosphate, there was minimal GTP hydrolysis. With all other organic anions, GTP hydrolysis substantially exceeded polymerization at all time points, with the onset of hydrolysis significantly preceding the onset of turbidity development. Nevertheless, the rate of GTP hydrolysis was a sigmoidal function of tubulin concentration under all conditions examined, suggesting that tubulin-tubulin interactions are required for hydrolysis. All anion-induced reactions were temperature dependent and cold reversible, but only the creatine phosphate induced reaction was not inhibited by GDP, CA2+, or colchicine and did not require GTP.