Purification of ribosomal proteins from Escherichia coli by cation exchange and reversed phase FPLC.

A complex mixture of 21 proteins from the 30S ribosomal subunit of Escherichia coli was fractionated on a cation-exchanger, then further separated on a C8 reversed-phase column. A set of 14 proteins were purified to homogeneity. The same protein mixture was also analysed on a C8 RPC column using a triethylamine phosphate (TEAP, pH2.2)/acetonitrile or a trifluoroacetic acid/acetonitrile solvent system which gave 11 and 8 purified proteins, respectively. Altogether, 16 out of 21 proteins from the 30S ribosomal subunit were purified.

The effect of formycin B on mRNA translation and uptake of purine precursors in Leishmania mexicana.

Formycin B is a structural analog of inosine that is a potent inhibitor of Leishmania multiplication. Formycin B is reportedly converted to formycin A nucleotides and incorporated into RNA by the organisms, and it is unclear whether the active form of the drug is the nucleoside itself or its several metabolites. We confirmed that formycin A nucleotides are formed by formycin B-exposed L. mexicana promastigotes, and determined that the intraparasite concentration of Formycin B and its metabolites was 6 times the extracellular formycin B concentration. Formycin B did not significantly inhibit purine nucleoside transport by intact promastigotes or purine base phosphoribosylation by parasite lysates. Thus, the nucleoside does not appear to inhibit these initial steps of purine nucleoside metabolism. Since RNA and protein synthesis in formycin B-treated intact promastigotes was found to be inhibited within 30 minutes, the effect of formycin A metabolites on leishmanial protein synthesis was investigated in in vitro protein synthesis experiments. Messenger RNA from formycin B-treated promastigotes was translated only 40% as efficiently as control promastigote mRNA by rabbit reticulocyte lysates. In addition, when formycin A-5'-triphosphate was preincubated with the rabbit reticulocyte lysates, translation of control mRNA was 86% inhibited. Formycin B toxicity to Leishmania promastigotes appears to be at least partially due to inhibition of protein synthesis by formycin A nucleotides and formycin A containing mRNA.

Ribosomal proteins: their structure and spatial arrangement in prokaryotic ribosomes.

During the last 15 years of ribosomal protein study, enormous progress has been made. Each of the proteins from E. coli ribosomes has been isolated, sequenced, and immunologically and physically characterized. Ribosomal proteins from other sources (e.g., from some bacteria, yeast, and rat) have been isolated and studied as well. Several proteins have recently been crystallized, and from the X-ray studies it is expected that much important information on the three-dimensional structure will be forthcoming. Many other proteins can probably be crystallized if suitable preparative procedures and crystallization conditions are found. Tremendous progress has also been made in deciphering the architecture of the ribosome. A battery of different methods has been used to provide the nearest neighbor distances of the ribosomal proteins in situ. Definitive measurements are now emanating from neutron-scattering experiments which also promise to give reasonably accurate radii of gyration of the proteins in situ. In turn, refined immune electron microscopy results supplement the neutron-scattering data and also position the proteins on the subunits themselves. This cannot be done by the other methods. Determination of the three-dimensional RNA structure within the ribosome is still in its infancy. Nonetheless, it is expected that by combining the data from protein-RNA and from RNA-RNA cross-linking studies, the structure of the RNA in situ can be unraveled. Of great interest is the fact that ribosomal subunits and ribosomes themselves have now been crystallized, and low-resolution structural maps have already been obtained. However, to grow suitable crystals and to resolve the ribosomal structure at a sufficiently high resolution remains a great challenge and task to biochemists and crystallographers.

Physical studies on the ribosomal protein S2 from the Escherichia coli 30S.

The protein S2 has been isolated from the 30S subunit of Escherichia coli A19 ribosomes [Littlechild, J., & Malcolm, A.L. (1978) Biochemistry 17, 3363-3369]. This salt-extracted protein is soluble and does not aggregate at salt concentrations of 0.3-0.4 M as used under reconstitution conditions. This differs from the S2 protein extracted by the acetic acid and urea method. The molecular weight from sedimentation equilibrium was found to be 29 200, and the protein was found to have a S0(20,w) value of 2.36S. The apparent specific volume at 20 degrees C was 0.726 mL.g(-1), and the D0(20,2) was 7.37 x 10(-7) cm(2)s(-1). The value for intrinsic viscosity was found to be 6.42 mL.g(-1). An axial ratio of (5-6):1 for a prolate ellipsoid of revolution was estimated by using these parameters. The circular dichroism and proton magnetic resonance studies show that protein S2 has both substantial amounts of alpha helix and beta-pleated sheet in solution and appears as a "folded" protein and not a random coil structure.

Physical studies on assimilatory nitrate reductase from Chlorella vulgaris.

Assimilatory nitrate reductase (EC 1.6.6.1 NADH:nitrate oxidoreductase) from Chlorella vulgaris purified by affinity chromatography was found to be homogeneous as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and by analytical ultracentrifugal techniques. The molecular weight of the intact enzyme and that of the enzyme dissociated in 6 M GuHCl, determined by sedimentation equilibrium studies, were 280,000 +/- 10,000 and 90,000 +/- 5,000, respectively. Comparable values were obtained using the S20,w value and the D20,w values in Svedberg's equation. The D20,w values were determined by laser light-scattering measurements. Active enzyme centrifugation showed that the monomer is an active species. A quantitative re-evaluation of the prosthetic groups present (FAD, heme, and molybdenum) was also made and was consistent with the conclusion that the active monomer contains three subunits as previously deduced by Solomonson et al. ((1975) J. Biol. Chem. 250, 4120). Electron micrographs showed images which corresponded to three subunits, supporting the data obtained by hydrodynamic studies. The enzyme is not cigar-shaped, as previously surmised, but has a roughly globular structure.

Shapes of proteins L1, L9, L25, and L30 from the 50S subunit of the Escherichia coli ribosome, determined by hydrodynamic studies.

Proteins L1, L9, L25, and L30, purified by a nondenaturing method from the 50S ribosomal subunit of Escherichia coli A19, have been characterized. The four proteins were studied under conditions which resemble those used for reconstitution experiments. These proteins have S020,W values of 2.0 S, 1.8 S, 1.8 S, and 1.0 S and D20,W values of 8.4 X 10(-7), 9.0 X 10(-7), 14.0 X 10(-7), and 15.0 X 10(-7) cm2/S. Apparent specific volumes at 20 degrees C are 0.738, 0.733, 0.700, and 0.735 mL/g for the four proteins. The respective molecular weights determined by sedimentation equilibrium are 25 000, 17 300, 12 000, and 6500. The intrinsic viscosity values for the four proteins are 4.0, 5.5, 3.6, and 3.2 mL/g. From these hydrodynamic parameters L1 and L9 appear to have globular or at most only slightly elongated shapes, whereas L25 and L30 appear to be definitely globular.

Shape of proteins S3 and S17 from the small subunit of Escherichia coli ribosome.

The shapes of proteins S3 and S17 purified from the 30 S subunit of Escherichia coli A19 were studied by hydrodynamic methods. The proteins have s020,w values of 2.1 +/- 0.1 S and 1.2 +/- 0.1 S and D020,w values of 7.4 +/- 0.5 . 10(-7) cm2/s and 11.4 +/- 0.6 . 10(-7) cm2/s. The respective molecular weights determined by sedimentation equilibrium are 25 800 +/- 500 and 9900 +/- 300. The intrinsic viscosity values for the two proteins are 5.8 +/- 0.3 ml/g and 4.2 +/- 0.2 ml/g. From these hydrodynamic parameters a slightly elongated shape for S3 and a globular shape for S17 have been concluded.

Shape of protein L11 from the 50S ribosomal subunit of Escherichia coli.

Protein L11 from the 50S ribosomal subunit of Escherichia coli A19 was purified by a method using nondenaturing conditions. Its shape in solution was studied by hydrodynamic and low-angle x-ray scattering experiments. The results from both methods are in good agreement. In buffers similar to the ribosomal reconstitution buffer, the protein is monomeric at concentrations up to 3 mg/mL and has a molecular weight of 16 000-17 000. The protein molecule resembles a prolate ellipsoid with an axial ratio of 5-6:1 a radius of gyration of 34 A, and a maximal length of 150 A. From the low-angle x-ray diffraction data, a more refined model of the protein molecule has been constructed consisting of two ellipsoids joined by their long axes.

Physical characteristics of protein-deficient derived from the 50S ribosomal subunit.

Sedimentation coefficients, diffusion coefficients, density increments, extinction coefficients, and molecular weights were determined for selected core particles from the 50S Escherichia coli ribosomal subunits. These core particles were found to be loosened structures containing less than half the protein present on the intact 50S subunits and totally lacking peptidyl transferase and polyphenylalanine synthesis activity unless reconstituted. The results of this study confirm that a loosening of the subunit takes place upon the removal of protein, but this loosening is not necessarily due to the loss of the protein.