Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Labuda, Damian; Janowicz, Zbigniew A.; Haertlé, Thomas; Augustyniak, J.

doi:10.1093/nar/1.12.1703

Cited by 9 publications

(5 citation statements)

References 18 publications

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“…In addition, it was recently demonstrated that the hexanucleotide containing fluorescent base and excised from barley tRNAPhe has a primary structure [25] identical to that reported for the anticodon region in other eukaryotic tRNAPhe already sequenced [26]. Since fluorescence changes observed for the two forms of barley phenylalanine tRNA, t R N A y and tRNA,Ph" [24], did not differ, we refer our results to barley tRNAPhe. The reported data thus comprise results obtained for both forms of barley tRNAPh".…”

supporting

confidence: 70%

“…This indicates that the whole primary structure is required to form structures characterised by maximal fluorescence intensity of intact tRNAPhe. Uncorrected fluorescence spectra of barley embryo tRNAPh' are very similar to those measured for other eukaryotic tRNAPh's [24]. In addition, it was recently demonstrated that the hexanucleotide containing fluorescent base and excised from barley tRNAPhe has a primary structure [25] identical to that reported for the anticodon region in other eukaryotic tRNAPhe already sequenced [26].…”

supporting

confidence: 61%

“…Barley embryo t R N A y and t R N A y were obtained as previously described [24,27]. Salt-free tRNA solutions were prepared by dialysis against Na2EDTA solutions followed by quartz-distilled water [28].…”

Section: Methodsmentioning

confidence: 99%

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Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

Labuda

Haertlé

Augustyniak

1977

European Journal of Biochemistry

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Barley phenylalanine tRNA (tRNAPhe) exhibits a 4.7-fold enhancement of fluorescence intensity in response to the addition of MgClz in 0.01 M Tris-HC1, pH 7.5,O.OOl M Naz EDTA. In the case of 2.0 M NaCl about 60% of this effect was observed. The change of fluorescence intensity was used as a measure of bound ligand concentration. From fluorescence Scatchard plots a different character of Mg2+ binding to tRNA was revealed, depending upon the initial conformation of tRNA in solution submitted to titration. In NaC1-free tRNAPhe solution strong interacting (K, = 1.5 x lo5 M-', n = 2.4) and weak independent (K, = 9 x lo3 M-') Mg2+ binding were observed. In solution containing 0.1 M NaCl both the strong and the weak Mg2+ binding demonstrated independent behaviour. K, and K,, in the presence of NaCl, had lower values than in NaC1-free solution, since Na+ was found to be a competitive inhibitor of M$+ binding. The significance of cations for tRNA conformation and biological activity is well established by studies of divalent cation binding to tRNA and of their effect on tRNA structure in solution ([l], for reviews see [2,3]). Recently the conformational effects of monovalent cations have been also well documented [3 -101. Less attention has been given to the simultaneous effect of mono and divalent cations on the tRNA molecule in solution, although studies of this effect give closer approximation of the applied experimental conditions to the physiological ones. From results obtained by Goldstein et al. [3] it could be assumed that the differences between the conformational effect of Na' and Mgz+ are mainly of a quantitative character. However, as recently shown [ l l ] the simultaneous effect of Na+ and M$+ on tRNA is not a simple sum of the effects of these ions observed separately. Studies of divalent cation binding to tRNA were performed by equilibrium dialysis [12,13] and other methods [13 -161 allowing for direct determination of bound and free ligand concentration at equilibrium conditions. Using these 'direct methods' the following parameters describing the binding process were obtained : the number of classes of binding sites and their independent or interacting character, the binding constant and number of binding sites within a class of binding sites. On the other hand, measurements of changes in some properties of macromolecule (e.g. fluorescence intensity) as a function of ligand concentration also reveal information concerning ligand binding to the macromolecule. Assuming that 100 % of the observed parameter change, at ligand concentration approaching infinity, reflects saturation of macromolecule with ligand, it is possible to determine the fraction of the macromolecules with occupied binding sites at any given ligand concentration. These methods of ligand binding measurements, which are called here 'indirect methods' have been already applied for studies of cation binding to tRNA [9]. Applying indirect methods, we measure the effect of ligand binding and thus, the observations are limited to those binding...

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confidence: 70%

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confidence: 61%

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Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

Labuda

Haertlé

Augustyniak

1977

European Journal of Biochemistry

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“…This would imply the presence of two different prosthetic groups which was never found in preparations of the apoenzyme. A reasonable tentative explanation is the presence of nucleic acids, which are known to emit (an emission ascribable to excimers [47]) in the wavelength region concerned [47,53,54]. The 3000-cm-' blue shift observed on going to glassy solution, which restricts the mobility of chromophores, would fit into an excimer picture.…”

Section: Luminescence Spectramentioning

confidence: 99%

On the Enigma of Old Yellow Enzyme's Spectral Properties

Eweg

Müller

Berkel

1982

European Journal of Biochemistry

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Old yellow enzyme (NADPH oxidoreductase) in the free and complexed state was thoroughly investigated by the following techniques : absorption, circular dichroism, fluorescence/phosphorescence and electron paramagnetic resonance spectroscopy and fluorescence/phosphorescence decay measurements, applied over a wide range of temperature (7 -293 K). The data obtained were interpreted by comparison with results from similar measurements on free FMN, existing spectral data on isoalloxazine model systems and theoretical data.The results clearly demonstrate the inadequacy of a simple phenolate-FMN donor-acceptor charge-transfer complex to explain the phenomena occurring upon the addition of phenols to old yellow enzyme. Instead it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably an H-bonded structure in which FMN is tautomerized and interacts with an L-chiral center. This is concluded from a separate electronic transition with an origin at 496 nm, thus far not recognized as such, and the circular dichroism observed.The emission is dominated by that of free FMN, although protein-bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon excitation of both native and complexed old yellow enzyme in the ultraviolet. This emission is quenched by the addition of phenol to the enzyme, shows a large (3000-cm-') blue shift on going to a low-temperature glass and is tentatively assigned to excimers of nucleic acids. Long-wavelength excitation with a synchronously pumped, mode-locked Rhodamine 6-G dye laser revealed a third, extremely weak emission in both native old yellow enzyme and its complexes. It decays with a lifetime of about 3 ns at 143 K. Electron paramagnetic resonance spectra revealed the presence of a low amount of an unpaired spin in old yellow enzyme. Owing to an unusual relaxational behaviour it could only be observed below 15 K and, again, the signal was measured in both the native enzyme and its complexes. Possible assignment and consequences of this observation are discussed.In frozen aqueous solutions of the enzyme-phenolate complex, a phase transition was discovered at which the colour of the complex reverted to that of the native enzyme. Subsequent melting restored the original colour.The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge is that of a very complicated network of hydrogen-bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long-wavelength absorption band in old yellow enzyme.The spectral changes induced by the binding of a lowmolecular-weight compound to old yellow enzyme (NADPH oxidoreductase) were initially regarded to be sufficiently mysterious to refer to this unknown compound as 'regreening factor' [I]. After the identification of this 'regreening factor'...

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“…It was shown to be pure and homogenous by two- To determine whether a Y-type base was present or not, fluorescence emission spectra (when excited at 310 nm) of the purified bean chloroplast tRNA Phe, of bean cytoplasmic tRNA Phe, of lupin (cytoplasmic) tRNA Phe, of yeast (cytoplasmic) tRNA Phe and of E. coli total tRNA were compared. As shown on fig.4, the fluorescence characteristic of the Y bases present in yeast, bean and lupin cytoplasmic tRNAs Phe is absent in bean chloroplast tRNA~ e. It should be noted that the fluorescence spectrum of bean cytoplasmic tRNA Phe is slightly different from that of yeast tRNA Phe, but identical to that of lupin tRNA Plae which is known to contain a peroxy-Y base [26], as in the case of wheat-germ [27] and barley [15,28] tRNAs Phe.…”

Section: Resultsmentioning

confidence: 99%

Purification and base composition of a chloroplastic tRNA^Phe from Phaseolus vulgaris

Guillemaut¹,

Martin²,

Weil³

1976

FEBS Letters

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Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Cited by 9 publications

References 18 publications

Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

On the Enigma of Old Yellow Enzyme's Spectral Properties

Purification and base composition of a chloroplastic tRNA^Phe from Phaseolus vulgaris

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Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Cited by 9 publications

References 18 publications

Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

On the Enigma of Old Yellow Enzyme's Spectral Properties

Purification and base composition of a chloroplastic tRNAPhe from Phaseolus vulgaris

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Product

Resources

About

Purification and base composition of a chloroplastic tRNA^Phe from Phaseolus vulgaris