Heavy and light forms of some aminoacyl-tRNA synthetases in fraction X,microsomes and cytosol of rabbit liver

Summary Aminoacyl-tRNA synthetase activity for alanine, glutamic acid, lysine and phenylalanine was studied in the three subcellular fractions of rabbit liver: fraction X, microsomes and cytosol. From 60 to 80% of the enzyme activities were found in fraction X and microsomes. Fraction X was especially rich in the synthetase activities. By means of gel chromatography, heavy (over 10⁶ daltons) and light (below 480 × 10³ daltons) forms of lysyl- and phenylalanyl- but only light ones of alanyl- and glutamyl-tRNA synthetase activities were found in all the subcellular fractions studied. It is concluded that in higher organisms (mammals) all aminoacyl-tRNA synthetases, at least in part, are associated with cell structural constituents.Abbreviations ALA, GLU, LYS, PHE alanyl-, glutamyl-, lysyl-, phenylalanyl-tRNA synthetase - PMSF phenylmethylsulfonyl fluoride - BSA bovine serum albumin     

Enhanced nucleotide analysis enables the quantification of deoxynucleotides in plants and algae revealing connections between nucleoside and deoxynucleoside metabolism

Detecting and quantifying low-abundance (deoxy)ribonucleotides and (deoxy)ribonucleosides in plants remains difficult; this is a major roadblock for the investigation of plant nucleotide (NT) metabolism. Here, we present a method that overcomes this limitation, allowing the detection of all deoxy- and ribonucleotides as well as the corresponding nucleosides from the same plant sample. The method is characterized by high sensitivity and robustness enabling the reproducible detection and absolute quantification of these metabolites even if they are of low abundance. Employing the new method, we analyzed Arabidopsis thaliana null mutants of CYTIDINE DEAMINASE, GUANOSINE DEAMINASE, and NUCLEOSIDE HYDROLASE 1, demonstrating that the deoxyribonucleotide (dNT) metabolism is intricately interwoven with the catabolism of ribonucleosides (rNs). In addition, we discovered a function of rN catabolic enzymes in the degradation of deoxyribonucleosides in vivo. We also determined the concentrations of dNTs in several mono- and dicotyledonous plants, a bryophyte, and three algae, revealing a correlation of GC to AT dNT ratios with genomic GC contents. This suggests a link between the genome and the metabolome previously discussed but not experimentally addressed. Together, these findings demonstrate the potential of this new method to provide insight into plant NT metabolism.

A new method for the quantification of (deoxy)ribonucleotides and nucleosides enables an in-depth analysis of the nucleotide metabolism in plants.     

Metal-enhanced fluorescence of tryptophan residues in proteins: Application toward label-free bioassays

The detection of submonolayers of proteins based on native fluorescence is a potentially valuable approach for label-free detection. We have examined the possibility of using silver nanostructures to increase the emission of tryptophan residues in proteins. Fluorescence spectra, intensities, and lifetimes of multilayers and submonolayers of proteins deposited on the surfaces of silver island films were measured. Increased fluorescence intensities from two- to three-fold and similar decreases in lifetimes were observed in the presence of the silver nanoparticles compared with the proteins on the surface of the bare quartz. The observed spectral effects of silver nanoparticles on tryptophan fluorescence indicates the possibility for the design of analytical tools for the detection of proteins without traditional labeling by extrinsic fluorophores.     

t-RNA-nucleotidyltransferase activity in Lupinus luteus seeds

t-RNA-nucleotidyltransferase activity was detected in Lupinus luteus seed.The enzyme was partly purified, and some of its properties are described.     

Sodium Green as a Potential Probe for Intracellular Sodium Imaging Based on Fluorescence Lifetime

We characterized the use of the fluorescent probe Sodium Green for measurements of intracellular free sodium using frequency–domain, phase–modulation fluorometry. The intensity decays were found to be strongly Na⁺dependent, with mean lifetime increasing from 1.13 ns in the absence of Na⁺to 2.39 ns in the presence of 140 mmNa⁺. Detailed analysis of the intensity decays in the presence of Na⁺and K⁺in the concentration range from 0 to 500 mmis provided. Sodium sensing using data measured at a single modulation frequency is described. Phase and modulation data showed high sensitivity to Na⁺and substantially lower sensitivity to K⁺. Additionally, exposure of Sodium Green to intense illumination indicated that Sodium Green is much more photostable than its precursor, fluorescein. These results indicate that lifetime-based measurements with Sodium Green can be used for imaging of intracellular free [Na⁺] in the range from about 0.5 to 50 mmwith high accuracy.     

Viscosity and sedimentation study of sonicated DNA–proflavine complexes

The intrinsic viscosity of sonicated calf thymus DNA (molecular weight 4–5 × 10⁵) increases and the sedimentation constant decreases, with increasing binding of proflavine at 0. 2 ionic strength and at 25°C. The measurements correspond to a linear increase in length of the almost rodlike DNA molecules with the amount of proflavine bound; independent calculations from viscosity and sedimentation measurements yield almost identical results. Over the range of r (moles of proflavine bound per moles of nucleotides) equal to zero to r = 0.13, the length increases by about 20%. This extension is compatible with the intercalation hypothesis proposed by Lerman. Density increments at various values of r, at constant chemical potential of diffusible solutes, were determined. It was also found that, in addition to the known isosbestic point of DNA-proflavine complexes at 455.5 mμ, an additional isosbestic point exists at 225.5 mμ; this proved extremely useful for the evaluation of binding studies.