The role of enzyme isomerization in the native catalytic cycle of the ATP sulfurylase-GTPase system.

ATP sulfurylase, from E. coli Kappa-12, is a GTPase.target complex that conformationally couples the free energies of GTP hydrolysis and activated sulfate (adenosine 5'-phosphosulfate, or APS) synthesis. Energy coupling is achieved by an allosterically driven isomerization that switches on and off chemistry at specific points in the catalytic cycle. This coupling mechanism is derived from the results of model studies using analogue complexes that mimic different stages of the native catalytic cycle. The current investigation extends the analogue studies to the native catalytic cycle. Isomerization is monitored using the fluorescent, guanine nucleotide analogues mGMPPNP (3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-[beta, gamma-imido]triphosphate) and mGTP [3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-triphosphate]. The isomerization is shown to be initiated by an allosteric interaction that requires the simultaneous occupancy of all three substrate-binding sites. Stopped-flow fluorescence and single-turnover studies were used to define and quantitate the isomerization mechanism, and to show that the isomerization precedes and rate-limits both GTP hydrolysis and APS synthesis. These findings are incorporated into a model of the energy-coupling mechanism.

Nucleotide hydrolysis-dependent conformational changes in p21(ras) as studied using ESR spectroscopy.

We have employed ESR spectroscopy using guanine nucleotides that contain a spin label at the 2',3'-position of the ribose to investigate structural changes in the proto-oncogene product p21(ras) that are dependent on nucleotide hydrolysis. The three nucleotide analogs used were 2',3'-(2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester (SL) GTP, SL-GDP, and the non-hydrolyzable analog SL-guanylylimidodiphosphate. SL-GTP was hydrolyzed by p21 with rates similar to those for GTP hydrolysis and appears to be an excellent substrate analog. The ESR spectra of SL-GTP and SL-GDP in complex with p21 differ significantly when acquired at 0 degrees C or 5 degrees C indicating different environments (conformations) of the protein-bound radicals depending on the phosphorylation state of the bound nucleotide. We calculated the rate constant for the conformational change as deduced from the changes in the corresponding ESR spectra upon incubation of the p21.SL-GTP complex at 25 degrees C and compared it to the rate constant of hydrolysis of SL-GTP at the same temperature. The rate constant deduced from the ESR method was similar to that determined by a high performance liquid chromatography technique. The data are in agreement with the idea that a conformational change during GTP hydrolysis by p21 occurs simultaneously with the actual hydrolysis step.

Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins.

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

Chromium(III) beta, gamma-bidentate guanine nucleotide complexes as probes of the GTP-activated cGMP cascade of retinal rod outer segments.

The exchange-inert Cr(III) beta, gamma-bidentate guanine nucleotide complexes Cr(III)GTP and Cr(III)Gpp(NH)p were used to probe the role of transducin in activating the retinal cGMP cascade. The Cr(III) nucleotide complexes were found to have lower binding affinity for transducin as compared to the Mg2+ complexes. However, the rate of hydrolysis of the transducin-bound Cr(III)GTP was similar to that of Mg(II)GTP. Cr(III)Gpp(NH)p activated the cGMP phosphodiesterase of photolyzed rod outer segment membranes up to 75% of the Mg(II)Gpp(NH)p level but lacked the ability to dissociated the transducin subunits from the rod outer segment membrane. This result implies that the activation of the phosphodiesterase by transducin-GTP complex is a membrane-associated event and the formation of a soluble complex of transducin-GTP with the inhibitory peptide of the phosphodiesterase may not be an obligatory step. Both the delta and lambda screw sense stereoisomers of Cr(III)Gpp(NH)p were capable of activating the cGMP cascade with no apparent stereoselectivity. The nature of the interaction of the metal ion and GTP at the nucleotide-binding site of transducin is discussed together with the results from previous studies using the phosphorothioate GTP analogues [Yamanaka, G., Eckstein, F., & Stryer, L. (1985) Biochemistry 24, 8094-8101] and is compared to the site found in homologous GTP-binding proteins such as elongation factor Tu [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36; la Cour, T.F.M., Nyborg, J., Thirup, S., & Clark, B.F.C. (1985) EMBO J. 4, 2385-2388]. The implications of the observed results on the molecular mechanism of visual signal transduction are discussed.

Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel currents in sensory neurones.

The influence of guanine nucleotide analogues on calcium channel currents in cultured rat dorsal root ganglion neurones has been studied using a technique in which the rate of diffusion of the analogues to their site of action is by-passed by photochemical release of the analogues within the neurones. The 1(2-nitrophenyl)ethyl P3-ester derivatives of guanosine 5'-0(3-thio)triphosphate (caged GTP-gamma-S) and 5'-guanylylimidodiphosphate (caged GMP-PNP) were synthesised and found to be completely photolysable by light, yielding free GTP-gamma-S and GMP-PNP. Calcium channel currents were recorded using the whole cell patch technique and either caged GTP-gamma-S or caged GMP-PNP (2 mM) were included in the patch pipette. Stable currents were recorded for 5-10 min, and a single pulse of 300-350 nm irradiation was directed using a liquid light guide onto the recording dish. Calcium channel currents were then recorded every 30-120 s following photochemical release of approximately 20 microM GTP-gamma-S. The peak calcium channel current was reduced by about 70% with a slow time course [t1/2 1.5 +/- 0.2 min (mean +/- SEM); n = 5]. The transient component of the peak current was usually completely abolished, whereas the sustained current measured at the end of the 100 ms depolarising pulse was less affected. Qualitatively similar effects were observed on photolysis of caged GMP-PNP.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of biodistribution of 3H, 32P and 99mTc labeled Gpp(NH)p in tumor bearing hamsters.

Biodistribution and tumor uptake studies were carried out with intravenously injected tracer doses of Gpp(NH)p labeled with 3H, 32P or 99mTc . Syrian golden hamsters with cheek pouch carcinomas, induced by repeated topical applications of DMBA, were used as a tumor model. The biodistributions of these three radionuclides were different, indicating significant molecular cleavage of this nucleotide analog. It was also apparent that this compound labeled with 99mTc may not be useful for tumor imaging due to low tumor-to-blood specific activity ratio. The cheek pouch carcinoma tumor model may be valuable for the evaluation of tumor localizing radiopharmaceuticals.