Ultraviolet absorption spectra of adenosine-5′-triphosphate and related 5′-ribonucleotides

Bock, Robert M.; Ling, N. R.; Morell, Samuel A.; Lipton, S. H.

doi:10.1016/0003-9861(56)90123-0

Cited by 327 publications

(100 citation statements)

References 8 publications

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“…The final G t concentration ranged between 30 and 40 M. To determine the amount of activatable G t , G t was titrated with exact amounts of GTP␥S (250 nM) in the presence of 500 nM R* under constant stirring in a final volume of 700 l (40). GTP␥S concentration was determined by UV/ visible absorption spectroscopy with 253 ϭ 13,700 M Ϫ1 ⅐cm Ϫ1 (41). All calculated activation rates in this work refer to the amount of activatable G t as determined by GTP␥S titration.…”

Section: Construction Of R-mvenus R-mecfp and Mvenus-mecfp Fusionmentioning

confidence: 99%

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Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

Ernst

Gramse

Kolbe

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

200

163

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G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (G␣␤␥).Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with G␣␤␥. To investigate whether a G protein-coupled receptor monomer can activate G␣␤␥, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G t) to determine the stoichiometry of rhodopsin/Gt binding and the rate of catalyzed nucleotide exchange in G t. Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G t was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G t interaction yielded a maximum of Ϸ50 Gt molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of Gt with an activated rhodopsin monomer is sufficient for fully functional Gt activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated. enzyme catalysis ͉ nucleotide exchange ͉ light scattering

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Section: Construction Of R-mvenus R-mecfp and Mvenus-mecfp Fusionmentioning

confidence: 99%

“…SEC runs with R*/Gt mixtures were performed with detection at 253 nm to monitor GDP ( 253 ϭ 13,700 M Ϫ1 ⅐cm Ϫ1 ; ref. 41) and 380 nm to monitor R* ( 380 ϭ 42,000 M Ϫ1 ⅐cm Ϫ1 , see above). Release of one molecule of GDP per R*⅐G t complex is given by the ratio of peak areas:…”

Section: Molar Extinction Coefficients For Analysis Of Uv/visible Absmentioning

confidence: 99%

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

Ernst

Gramse

Kolbe

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

200

163

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“…89 The concentrations of ATP and ATP©S (both from Roche) were determined using the extinction coefficient 15,400 M −1 ·cm −1 at 259 nm. 90 The concentrations of NADH and phosphoenolpyruvate (both from Sigma) were determined using the extinction coefficients 6220 M −1 cm −1 at 340 nm and 2930 M −1 cm −1 at 230 nm, respectively. Lactic dehydrogenase and pyruvate kinase were obtained from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

Singleton¹,

Roca²,

Lee³

et al. 2007

Tetrahedron

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The RecA protein of Escherichia coli plays a crucial roles in DNA recombination and repair, as well as various aspects of bacterial pathogenicity. The formation of a RecA-ATP-ssDNA complex initiates all RecA activities and yet a complete structural and mechanistic description of this filament has remained elusive. An analysis of RecA-DNA interactions was performed using fluorescently labeled oligonucleotides. A direct comparison was made between fluorescein and several fluorescent nucleosides. The fluorescent guanine analog 6-methylisoxanthopterin (6MI) demonstrated significant advantages over the other fluorophores and represents an important new tool for characterizing RecA-DNA interactions.

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“…The solvents used were 0.16 M LiCl or 0.5 M sodium formate pH 3.6 (PEI-cellulose), isopropanol : 25% NH3 : H20 = 6:3:1 (kieselgel thin layer plates) and 0.1 M sodium phosphate (pH 6.8) : (NH4)2S04 : n-propanol = 100 : 60 : 2 (3 MM paper) (Randerath and Randerath, 1967;Hanna and Meares, 1983;Bock et al, 1956). …”

Section: Isolation Of the Labelled Dinucleotide Apu From The B Subunimentioning

confidence: 99%

Affinity Labelling of the Active Center of DNA-dependent RNA Polymerases within the Archaebacterial Kingdom

Thomm¹,

Lindner²,

Hartmann³

et al. 1988

Systematic and Applied Microbiology

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SummaryThe super-selective affinity labelling procedure for the active center of RNA Polymerase from Escherichia coli (Grachev et al., 1987a) was successfully applied to the enzyme from archaebacteria. Using adenosine-5'-trimetaphosphate or the p-hydroxybenzaldehyde ester of ATP, the second largest subunit B' of the RNA polymerase from methanogenic/halophilic and sulphate reducing archaebacteria, and the largest subunit B of the non-methanogenic thermophilic archaebacterium Sulfolohus sp. strain B 12 are labelled specifically. The labelling reaction is strictly template-dependent and blocked by the transcription inhibitor heparin. We present evidence that adenosine-5'-trimetaphosphate is attached close to the catalytic center of the RNA polymerase via a phosphoamide bond to lys or his residues.The specific affinity labelling of subunit B' from methanogens/halophiles together with the earlier observed immunological cross-reactivity (Gropp et al., 1986) indicates that this subunit as well as subunit B" contains amino acid sequences which are homologous to sequences in subunit B of the enzyme from non-methanogenic thermophilic archaebacteria and in subunit ß from E. coli RNA polymerase.

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Ultraviolet absorption spectra of adenosine-5′-triphosphate and related 5′-ribonucleotides

Cited by 327 publications

References 8 publications

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

Affinity Labelling of the Active Center of DNA-dependent RNA Polymerases within the Archaebacterial Kingdom

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