PubMed · 6777900
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B A Fiedel, H Gewurz. Complement (Clq), polyanions and platelet activation.. https://doi.org/10.1016/0049-3848(80)90047-x
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When grown under phosphate-limiting conditions Pseudomonas aeruginosa expresses the phosphate-specific porin OprP. In order to determine whether any of the lysine residues located in the amino-terminal half of the protein play a role in the transport of anions through the channels, the first nine amino-terminal lysine residues of OprP were substituted with glutamates. The mutant proteins were purified and the channels they formed were characterized by reconstituting the purified porins in planar lipid membranes. In comparison to the wild-type protein, the Lys74, Lys121, and Lys126 mutants all displayed reduced levels of conductance at KCl concentrations below 1 M, and the Lys74 and Lys121 mutants no longer exhibited a saturation of conductance at high anion concentrations. In addition, the ability of phosphate ions to inhibit the conductance of Cl- ions through the channels formed by the Lys121 mutant was greatly reduced, while their ability to inhibit the Cl- conductance of the Lys74 mutant was reduced by approximately 2-fold. To clarify the roles that Lys74, Lys121, and Lys126 play in regulating the channel characteristics of OprP, these amino acids were replaced with either glycine or glutamine residues. Analysis of these mutants suggested that both Lys74 and Lys126 may serve to funnel anions toward the binding site, but only the presence of Lys121 is required for the formation of the inorganic phosphate-specific binding site of OprP.
The interaction of carnitine with human placental brush-border membrane vesicles was investigated. Carnitine was found to associate with the membrane vesicles in a Na(+)-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na+ gradient-driven transport process. The absolute requirement for Na+ was noticeable whether the association of carnitine with the vesicles was measured with a short time incubation or under equilibrium conditions, indicating Na(+)-dependent binding of carnitine to the human placental brush-border membranes. The binding was saturable and was of a high-affinity type with a dissociation constant of 1.37 +/- 0.03 microM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analyses revealed that Na+ increased the affinity of the binding process for carnitine and the Na+/carnitine coupling ratio for the binding process was 1. The dissociation constant for the interaction of Na+ with the binding of carnitine was 24 +/- 4 mM. This constitutes the first report on the identification of Na(+)-dependent high-affinity carnitine binding in the plasma membrane of a mammalian cell. Studies with purified rat renal brush-border membrane vesicles demonstrated the presence of Na+ gradient-driven carnitine transport but no Na(+)-dependent carnitine binding in these membrane vesicles. In contrast, purified intestinal brush-border membrane vesicles posses neither Na+ gradient-driven carnitine transport nor Na(+)-dependent carnitine binding.
Proteins of the transferrin family bind, with high affinity, two Fe3+ ions and two CO3(2)- ions but can also bind other metal ions and other anions. In order to find out how the protein structure and its two binding sites adapt to the binding of larger anions, we have determined the crystal structure of oxalate-substituted diferric lactoferrin at 2.4 A resolution. The final model has a crystallographic R-factor of 0.196 for all data in the range 8.0-2.4 A. Substitution of oxalate for carbonate does not produce any significant change in the polypeptide folding or domain closure. Both binding sites are perturbed, however, and the effects are different in each. In the C-lobe site the oxalate ion is bound to iron in symmetric 1,2-bidentate fashion whereas in the N-lobe the anion coordination is markedly asymmetric. The difference arises because in each site substitution of the larger anion causes displacement of the arginine that forms one wall of the anion binding site; the movement is different in each case, however, because of different interactions with "second shell" amino acid residues in the binding cleft. These observations provide an explanation for the site inequivalences that accompany the substitution of non-native anions and cations.