GTP Alpha Research Articles

Interaction of tubulin with guanosine 5'-O-(1-thiotriphosphate) diastereoisomers: specificity of the alpha-phosphate binding region.

The exchangeable nucleotide-binding site of tubulin has been studied using diastereoisomers A (Sp) and B (Rp) of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) in which the phosphorus atom to which sulfur is attached is chiral. GTP alpha S(A) (10 microM) nucleated assembly of purified tubulin (20 microM) into microtubules in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid with 3 mM Mg2+ and 1 mM EGTA, pH 6.6 at 37 degrees C. With 0.2 mM GTP alpha S(A), the critical concentration (Cc; minimum protein concentration required for assembly) was 8 microM tubulin. Neither 0.2 mM GTP nor GTP alpha S(B) promoted microtubule assembly in buffer with 0.5-6.75 mM Mg2+ and 20-70 microM tubulin. The Cc values for GTP alpha S-(A)-induced assembly of tubulin in buffer with 30% glycerol and of microtubule protein (tubulin and microtubule-associated proteins) in buffer were lower than for GTP. GTP alpha S(A)-induced microtubules were more stable to the cold and to Ca2+. GTP alpha S(A) and GTP but not GTP alpha S(B) bound tightly to tubulin at 4 degrees C. Although GTP alpha S(B) did not nucleate assembly, it did bind to tubulin since it was incorporated into the growing microtubule. Both isomers were hydrolyzed in the microtubules. These studies show that GTP alpha S(A) promotes tubulin assembly better than GTP and GTP alpha S(B) and that there is stereoselectivity at the alpha-phosphate binding region of tubulin. The stereoselectivity may be due to different MgGTP alpha S(A) and -(B) interactions with tubulin.

Nucleotide inhibitors and activators of retinal guanylyl cyclase.

The restoration of the dark state in retinal rod cells following illumination is due in part to resynthesis of cGMP. Retinal guanylyl cyclase specifically catalyzes the cyclization of GTP into cGMP in vivo. The reaction has been shown to involve the inversion of the configuration on the phosphate atom as demonstrated by conversion of the (SP) isomer of GTP alpha S to (RP)-cGMPS by guanylyl cyclase [Senter, P. D., Eckstein, F., Mülsch, A., & Böhme, E. (1983) J. Biol. Chem. 258, 6741-6745]. Since (RP-cGMPS is not a substrate for retinal phosphodiesterase, we were able to measure cyclase activity with greater reliability using this novel assay as opposed to other published procedures. This assay has allowed us to reinvestigate the effects of adenylyl nucleotides on cyclase activity and to search for selective inhibitors of the rod-specific enzyme. We have measured the cyclase activity using homogenates of rod outer segments and a reconstituted system composed of guanylyl cyclase in washed rod outer segment membranes and the purified guanylyl cyclase activating protein. Our results indicate that 100-200 microM ATP (and other adenylyl nucleotides) stimulates guanylyl cyclase activity approximately 2-fold and that the observed stimulation of enzyme activity is independent of the free calcium concentration. In contrast to other particulate guanylyl cyclases, which are synergistically stimulated by a peptide ligand and ATP, guanylyl cyclase activating protein does not potentiate the effect of ATP, suggesting that retinal guanylyl cyclase may be regulated differently. ATP changes the Vmax of retinal guanylyl cyclase without changing the Km for (SP)-GTP alpha S.(ABSTRACT TRUNCATED AT 250 WORDS)

GTP hydrolysis by purified .alpha.-subunit of transducin and its complex with the cyclic GMP phosphodiesterase inhibitor

The single-turn GTP hydrolysis by isolated and soluble transducin has been time-resolved using a rapid flow filtration technique which takes advantage of the GTP-requiring detachment of transducin alpha-subunits (T alpha) from photoactivated rhodopsin (R*). Illuminated rod outer segment (ROS) fragments to which holo-transducin is tightly bound are retained on a syringe filter that is washed continuously with a buffer containing no GTP. When the flow is switched to a buffer with GTP, T alpha GTP is specifically eluted and injected into a cuvette where GTP hydrolysis is monitored via the associated change in the T alpha intrinsic tryptophan fluorescence. Low concentrations of GTP elute the complete pool of T alpha from the filter-retained ROS fragments in less than 1 s. This directly demonstrates that, upon GTP loading, T alpha becomes instantly soluble in physiological buffers (120 mM KC1 and 2 mM MgCl2). When all alone, T alpha hydrolyzes its bound GTP in 21 +/- 1 s (1/e time at 25 degrees C). Replacing chloride by other anions increases the GTPase rate by 2-fold. The K50 for chloride inhibition of GTPase is approximately 2 mM. Slower GTP hydrolysis is observed for cholera-toxin-modified transducin or when GTP alpha S (Sp) replaces GTP in the eluting buffer. No signal is observed when GTP gamma S is used. The GTPase rate is unaffected when T alpha GTP binds to the inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE), although this binding is fast and of high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Low structural specificity for nucleoside triphosphates as antagonists of ADP-induced platelet activation.

Although the platelet ADP receptor is thought to exhibit a high degree of structural selectivity, adenosine 5'-O-(thiotriphosphate) (ATP alpha S) is a potent inhibitor of ADP-induced platelet activation and has been recently shown to bind with high affinity (Kd 3 +/- 0.1 nM) to formaldehyde-fixed platelets and to be photoincorporated into an 18-kDa fragment beginning at Tyr-198 of glycoprotein (GP) IIb alpha (Greco, N. J., Yamamoto, N., Jackson, B. W., Tandon, N. N., Moos, M., Jr., and Jamieson, G. A. (1991) J. Biol. Chem. 266, 13627-13633). Further studies have now shown that guanosine 5'-O-(thiotriphosphate) (GTP alpha S) also binds to high affinity sites (Kd 4.7 +/- 0.9 nM; 13,600 +/- 1,140 sites/platelet) and to low affinity sites (Kd 470 +/- 85 nM; 135,900 +/- 19,400 sites/platelet). Competition binding studies showed that all GTP alpha S binding sites were accessible to ADP and vice versa. The corresponding pyrimidine nucleotide cytidine 5'-O-(thiotriphosphate) (CTP alpha S) was found to be similarly effective in competing in the binding of ADP and both 5'-O-(thiotriphosphates) as well as uridine 5'-O-(thiotriphosphate) (UTP alpha S) were potent inhibitors of platelet shape change and aggregation. Ultraviolet irradiation of platelets in the presence of either [35S]GTP alpha S or [35S]UTP alpha S resulted in their specific incorporation into the alpha chain of GPIIb as previously shown with [35S]ATP alpha S. These results show that the structure of the nucleotide base has little influence on its ability to occupy the ADP-binding site on platelets, to function as an inhibitor of ADP-induced activation or to be photoincorporated into GPIIb alpha.

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments.

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.

Biochemical characterization of three stimulatory GTP-binding proteins. The large and small forms of Gs and the olfactory-specific G-protein, Golf.

The biochemical properties of three stimulatory guanine nucleotide-binding protein (G-protein) alpha subunits, the large and small forms of Gs, Gs-l (52 kDa) and Gs-s (45 kDa), and the olfactory specific G-protein, Golf, have been compared. Complementary DNAs (cDNAs) encoding each alpha subunit were independently expressed in a mammalian cell line deficient in endogenous stimulatory G-proteins (S49 cyc-kin-). Gs-l and Gs-s respond similarly to activation by the beta-adrenergic agonist isoproterenol (EC50 = 80 and 60 nM, respectively) and the receptor-independent G-protein activators guanosine 5-O-3-(thio)triphosphate) (GTP gamma S) and AlF-4. The ability of Golf to interact with the beta-adrenergic receptor was also examined. Surprisingly, Golf interacts with beta-adrenergic receptors and is activated by isoproterenol (EC50 = 120 nM). All three G-proteins respond similarly to treatment with different alpha, beta, and gamma thiophosphoryl analogs of GTP. Specifically, (R)-GTP alpha S and GTP gamma S activate each G-protein, whereas (S)-GTP alpha S and (R)- or (S)-GTP beta S are inactive. In addition, similar to Gs alpha, Golf alpha is covalently modified and constitutively activated by cholera toxin. These studies demonstrate that all three stimulatory G-proteins are functionally and structurally similar, however, subtle differences between Golf and the two forms of Gs appear to modulate their interactions with receptors.

Characterization of the elongation factors from calf brain. 2. Functional properties of EF-1 alpha, the action of physiological ligands and kirromycin.

The properties of EF-1 alpha from calf brain have been investigated and compared with those of EF-Tu. EF-1 alpha binds GDP and GTP in a 1:1 stoichiometry, showing the same affinity for both nucleotides (K'd = 2-4 microM). EF-1 beta strongly enhances the dissociation rate of the EF-1 alpha X GDP complex and to a lesser extent of the EF-1 alpha X GTP complex. Aminoacyl-tRNA (aa-tRNA) stabilized EF-1 alpha X GTP much less efficiently than the EF-Tu X GTP complex. Unlike EF-Tu, EF-1 alpha sustains the binding of aa-tRNA to the ribosome also in the presence of GDP or in the absence of any nucleotide, though to a lesser degree than with GTP. Kirromycin enhances the dissociation rate of both EF-1 alpha X GTP and EF-1 alpha X GDP but especially that of the latter. This effect results in an increase of the exchange rate of the EF-1 alpha-bound nucleotide with free nucleotides. Although in this regard the effect of kirromycin mimics that of EF-1 beta, the antibiotic is incapable of increasing the EF-1 alpha X GDP/GTP exchange rate when aa-tRNA and ribosomes are present. Therefore, unlike EF-1 beta, kirromycin cannot enhance the rate of poly(Phe) synthesis. On the other hand, the failure of kirromycin to induce a GTP-like conformation of EF-1 alpha X GDP, as in the case of EF-Tu X GDP, explains its inability to inhibit peptide bond formation in the eukaryotic system.

Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site.

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.

Guanosine thiophosphate derivatives as substrate analogues for phosphoenolpyruvate carboxykinase.

The interactions of nucleotides with phosphoenolpyruvate carboxykinase were studied by using the stereospecific thiophosphate analogues of GDP and GTP. The metal ion dependent stereoselectivity of these analogues was determined by using steady-state kinetics. The RP and SP isomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S) were substrates with low turnover, and a small preference for the RP isomer was observed. Neither the enzyme-metal nor the nucleotide-metal complex elicited any substantial change in the selectivity. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) exhibited no substrate activity for the enzyme, regardless of the cations. This nucleotide was a competitive inhibitor against GDP, however. Both RP and SP diastereomers of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) were good substrates for phosphoenolpyruvate carboxykinase; in several cases, depending upon the cation, kcat and/or Vm/Km for the RP isomer is greater than for the substrate GTP. The enzyme-metal complex but not the nucleotide-metal complex affects the relative Km and the Vmax values. In contrast, guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) (SP) is a much better substrate (greater than 50 times) than is GTP beta S (RP). The metal ions have little effect on the selectivity. These results suggest a specific interaction of the beta-phosphate of the nucleotide with the protein. The analogue guanosine 5'-O-(3-thiotriphosphate) (GPT gamma S) serves as a substrate to yield GDP and thiophosphoenolpyruvate. The latter was detected by 31P NMR and was shown to slowly hydrolyze to form phosphoenolpyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereochemistry of the guanyl nucleotide binding site of transducin probed by phosphorothioate analogues of GTP and GDP.

The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducin has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP gamma S = (Sp)-GTP alpha S greater than (Rp)-GTP alpha S greater than (Sp)-GTP beta S much greater than (Rp)-GTP beta S. The affinity of transducin for GTP gamma S is greater than 10(4) higher than that for (Rp)-GTP beta S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP alpha S with a l/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP alpha S, like GTP gamma S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP alpha S greater than GDP greater than (Rp)-GDP alpha S greater than GDP beta S. The affinity of transducin for (Sp)-GDP alpha S is about 10-fold higher than that for GDP beta S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the alpha- or beta-phosphorus atom of GTP. These results lead to the following conclusions: The pro-R oxygen atom at the alpha-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. The pro-S oxygen at the alpha-phosphorus does not appear to be involved in a critical interaction with transducin.(ABSTRACT TRUNCATED AT 250 WORDS)