Arginyl-tRNA synthetase from Bacillus stearothermophilus (NCA 151 8 ) has been purified approximately 450-fold to 9001, homogeneity. The enzyme has a molecular weight of 78000 as estimated by disc electrophoresis in denaturing conditions (dodecylsulfate). No subunits were revealed in these conditions. The K , values for arginine, ATP and tRNA have been determined. Like the arginyl-tRNA synthetase from other organisms, this one from B. stearothermophilus requires tRNA to catalyse an arginine-dependent ATP/[32P]PPi exchange reaction. Periodatetreated tRNA does not induce this exchange reaction. Aminoacylation is not completely inhibited by 2 mM pyrophosphate, indicating that the exchange reaction may be explained by the reversal of the overall amiiioacylation reaction.Different methods tested failed to reveal any arginyl-adenyla6e intermediary. By gel Gltration, however, enzyme-bound arginine could be isolated from a system containing all the substrates of the enzyme.The binding of arginine and ATP to arginyl-tRNA synthetase has been studied by equilibrium partition and shows that arginine is bound only after the binding of tRNA and ATP.The binding of ATP does not depend on the presence of other substrates in the reaction mixture. One binding site for arginine and one for ATP are found per molecule of active enzyme. Periodate-oxidized tRNA and phenoxyacetylarginyl-tRNA did not induce the binding of arginine to the enzyme. Intact tRNA from other species which could be aminoacylated by arginyltRNA synthetase from B. stearothermophilus, also induced the binding of arginine to the enzyme. Nucleotides other than ATP were not effective in the binding of arginine.It is concluded that the mechanism of action of this enzyme is a concerted one, as proposed by Loftfield and Eigner, in which arginine is bound after tRNA and ATP.Among all the known aminoacyl-tRNA synthetases, the ones specific for arginine, glutamate and glutamine differ from the others with respect to the ATP/[32P]PPi exchange reaction. The arginyl-tRNA synthetase from Escherichia coli [4,5] and from yeast

Ferritin was isolated from the seeds of pea (Pisum sativum) and lentil (Lens esculenta). The homogeneity of the phytoferritins was established by polyacrylamide-gel electrophoresis. The subunit molecular weights were respectively 20 300 and 21 400 for hte pea and lentil proteins. A neutron low-angle scattering study established the molecular weight of the oligomer as 480 000 for pea apoferritin and 510 000 for lentil apoferritin. Although the quaternary structure of 24 polypeptide chains is preserved, the phytoferritins have a larger cavity in the interior than mammalian ferritins and can thus potentially store 1.2-1.4 times as much iron. The amino acid composition of the phytoferritins show some similarities to those of mammalian apoferritins; tryptic 'fingerprinting' reveals that there are many differences in the amino acid sequence of plant and mammalian apoferritins.

Virginiamycin S (VS, a type B component of the synergistin group of antibiotics) is fluorescent in solution: the fluorescence intensity is proportional to VS concentration. The intensity of VS fluorescence was found to increase upon addition of 50S ribosomal subunits, and this variation (deltaI 416 nm) to be proportional to the concentration of 50S subunits. This new technique was, then, used to measure the binding reaction of VS to ribosomes. Similar patterns of linkage were obtained for ribosomes and large subunits, whereas very little fixation to 30S particles was detected. The binding reaction was virtually instantaneous at any temperature, and, for saturating VS, was not influenced by Mg++ concentration in the range 1 to 20 mM, nor by the replacement of 100 mM K+ with NH+4. The association constant of VS TO 50S particles was found to be KA=2.5 X 10(6)M-1, and from the Scatchard plot a v value of 0.9 was calculated, which points to a stoichiometric reaction leading to 1 mole VS bound per mole of 50S particles. Upon fixation of virginiamycin M (VM, a type A component of the synergistin group of antibiotics), the delta I of the VS-ribosome complex was increased, and a KA=15 x 10(6)M-1 was recorded for the association constant of VS to 50S particles. Such sixfold increase in the affinity of ribosomes for VS may account for the synergistic effect of the 2 virginiamycin components in sensitive bacteria.

Extrapolation of a series of low-angle neutron scattering curves to infinitely high contrast gives a scattering function IC(K) which is dependent on the shape of the solute molecule. For the 50S subunit of E. coil ribosomes, the first part of the structure determination by neutron scattering, namely the determination of the molecular shape from IC(K), is reported.The result is in good agreement with models of the SOS subunit determined by electron microscopy.With the advent of high-flux reactors, neutron scattering has become a powerful technique in the investigation of biological structure. The usefulness of neutrons in scattering methods is apparent, especially for structures that have chemically distinct components and exhibit pronounced intramolecular fluctuations of scattering density (e.g., ribosomes, lipoproteins, viruses, and chromatin).Neutron small-angle scattering curves in H20/D20 mixtures correspond to various views of the same molecule in solution. If the mean scattering density of the solute is close to that of the solvent (which is the situation at low contrast), intramolecular fluctuations of scattering density will predominantly contribute to the scattering curve. At higher contrasts, the scattering curve essentially depends on the overall shape of the solute particle.Extrapolation of a series of scattering curves obtained in different solvents to infinitely high contrast yields a scattering function IC(K) that is entirely dependent on the shape of the solute molecule. At the start of a structure determination, the evaluation of IC(K) is very helpful because the shape is described by a smaller number of parameters than the whole structure. Moreover, the subsequent determination of the detailed internal structure is then greatly facilitated by the boundary conditions given by the shape.In this paper we present the first part of the structure determination of the larger subunit of Escherichia coli ribosomes by neutron scattering-the determination of the molecular shape from IC(K). These results are compared with models of the 50S ribosomal subunit as determined by electron microscopy. METHODSThe preparation of the large ribosomal subunit from E. coli B (strain MRE600) used the methods described (1 olation to zero concentration, we measured a series of five concentrations from 5 to 25 mg/ml for each contrast. The sedimentation profiles of H20 and D20 samples were analyzed for homogeneity, and were reanalyzed after neutron scattering measurements in order to detect possible alteration of the material. The samples were found to be unchanged. The activity of the 50S preparations in poly(U)-directed polyphenylalanine synthesis in the presence of 30S particles according to Fahnestock et al. (2) The average measuring time was 15 min at each position. Transmission measurements were made for each sample and used to determine the exact H20 content. Two sets of data obtained from independent preparations were collected at different temperatures. The first set in TMK buffer was measured at room temp...

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