ABSTRACT
The complex formation between elongation factor Tu (EF-Tu) . GTP and valyl-tRNA Val 1 has been investigated using the small-angle X-ray scattering titration technique. The main species observed is a 1:1 complex with a stability constant log K greater than or equal to 6. The corresponding interaction between EF-Tu . GTP and non-aminoacylated tRNA appears to be much weaker with an estimated log K approximately equal to 4. The radius of gyration determined for the EF-Tu . GTP -- valyl-tRNA Val 1 complex is larger (R = 3.6 nm) than that of EF-Tu . GTP (R = 2.5 nm). Likewise, the maximum distance within this complex is larger (Dmax = 12.5 nm) than the one within EF-Tu . GTP (Dmax = 8.5 nm). These data as well as the p(r) curve are consistent with a multiellipsoid model for the complex. From this model it is indicated that the acceptor stem of tRNA is attached to EF-Tu and that the anticodon stem and loop protrude into the solution.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/metabolism , Peptide Elongation Factors/metabolism , RNA, Transfer, Amino Acyl/metabolism , Macromolecular Substances , Nucleic Acid Conformation , Peptide Elongation Factor Tu , Protein Conformation , X-Ray DiffractionABSTRACT
Equilibrium analysis of a model system for the in vivo reactions between penicillamine and Cu(I), the penicillamine-glutathione-Cu(I) system, indicates that in a certain concentration range the use of penicillamine as a drug will not disturb the normal Cu(I) metabolism. The equilibrium data required for this analysis were obtained by emf titrations on the Cu(I)-glutathione (H3A) and the Cu(I)-pencillamine (H2A) systems at 25 degrees C. in 0.5 M NaClO4 medium, using glass and copper amalgam electrodes; the data were analyzed first by various graphical methods and then by a general least squares computer program. The results show that mononuclear Cu(I) species Cu(HA)2 form in both systems with stability constants log beta 122 of 38.8 (glutathione) and 39.18 (penicillamine); in addition, the polynuclear Cu5A43- species forms in the penicillamine system and the mononuclear CuHA- species might form in the glutathione system. The results are discussed in relation to the therapeutic use of penicillamine as well as in relation to the toxic action of copper on living cells.
Subject(s)
Copper , Glutathione , Penicillamine , Chemical Phenomena , Chemistry , Copper/metabolism , Hydrogen-Ion Concentration , Kinetics , Mathematics , Models, BiologicalABSTRACT
Copper (II) ions successively induce dimers and tetramers of human serum albumin (L) when the Cu (II) concentration is extended beyond that of 200 muM. This is shown by emf titrations and by ultracentrifugation experiments. The emf titrations, which involve a new pH static method, were performed at 25 degrees, in a 0.5 M NaCIO4 medium at pH 6.59, using glass and copper amalgam electrodes. The total concentration of Cu(II) varied from 0.14 to 2.2 mM and the albumin concentration from 0.05 to 0.7 mM. In order to evaluate the formula of the main complexes, without using any a priori assumptions regarding their compositions, a detailed graphic procedure was used. The results, in the form of equilibrium constants for the main species, were refined by the use of a general least squares computer program. The experimental data are found to be consistent with the formation of the monomeric CuL, Cu5L, and Cu6L species and the dimeric Cu3L2, Cu4L, Cu6L, and Cu8L2 species. In addition, there is some indication for a minor species, most probably the Cu12L4 tetramer. The pH static results qualitatively agree with the findings obtained by ultracentrifugation. As indicated by distinct bands and their S-values, ultracentrifugation experiments show not only monomeric and dimeric species of albumin, but also tetrameric species. The polymerization of the albumin is reversible, since diglycyl-L-histidine, a peptide designed to mimic the Cu (II) transport site of albumin, depolymerizes the Cu (II)-albumin polymers.
