Purification and characterization of the fimbria F18ac (2134P) isolated from enterotoxigenic Escherichia coli (ETEC).

The adhesin F18ac purified on Sepharose CL 4B column chromatography and SDS-PAGE stained with Coomassie Blue and Western blotting using specific anti-F18ac serum presented one band of approximately 17kDa. Gold immunolabeling revealed that the adhesin F18ac has a fimbrial structure on the bacterial surface. The first 27 amino acid residues of the N-terminal portion of the adhesin F18ac, showed 92.5% homology (25 amino acids) with the F107 (F18ab) fimbriae.

ATP-induced tetramerization and cooperativity in hemoglobin of lower vertebrates.

The importance of intraerythrocytic organic phosphates in the allosteric control of oxygen binding to vertebrate hemoglobin (Hb) is well recognized and is correlated with conformational changes of the tetramer. ATP is a major allosteric effector of snake Hb, since the absence of this nucleotide abolishes the Hb cooperativity. This effect may be related to the molecular weight of about 32,000 for this Hb, which is compatible with the dimeric form. ATP induces a pH-dependent tetramerization of deoxyHb that leads to the recovery of cooperativity. This phenomenon may be partially explained by two amino acid replacements in the beta chains (CD2 Glu-43 --> Thr and G3 Glu-101 --> Val), which result in the loss of two negative charges at the alpha1beta2 interface and favors the dissociation into dimers. The ATP-dependent dimer left arrow over right arrow tetramer may be physiologically important among ancient animal groups that have similar mutations and display variations in blood pH that are governed by these animals' metabolic state. The enormous loss of free energy of association that accompanies Hb oxygenation, and which is also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the transition from a myoglobin-like protein to an allosteric one may be of evolutionary significance.

Tobacco mosaic virus disassembly by high hydrostatic pressure in combination with urea and low temperature.

We investigated the effect of low temperature and urea combined with high pressure on tobacco mosaic virus (TMV). The evaluation of its aggregation state and denaturation process was studied using gel filtration, transmission electron microscopy, and spectroscopic methods. The incubation at 2.5 kbar induced 18% dissociation, and decreasing of temperature to -19 degreesC promoted additional dissociation to 72%, with stabilization of the dissociation products. Under such conditions, extensive denaturation did not occur. The apparent enthalpy and entropy of dissociation (Delta and TDelta) were -9.04 kcal/mol subunit and -15.1 kcal/mol subunit, respectively, indicating that the TMV association is an entropicly driven process. The apparent free energy of stabilization given by the presence of RNA is at least -1.7 kcal/mol subunit. Urea-induced dissociation of TMV samples and incubation at high-pressure promoted a higher degree of dissociation. The volume change of dissociation decreased in magnitude from -16.3 to -3.1 mL/mol of dissociated subunit, respectively, in the absence and presence of 2.5 M urea, suggesting exposure of the protein-protein interface to the solvent. High-pressure induced remarkable TMV denaturation in the presence of 2.5 M urea, with a volume change of -101 mL/mol of denatured subunit. The apparent enthalpy and entropy of denaturation (Delta and TDelta) by 1.75 M urea at 2.5 kbar was -11.1 and -10.2 kcal/mol subunit, respectively, demonstrating that the TMV protein coat presents an apparent free energy of denaturation by urea close to zero. Although the processes could not be assumed to be pure equilibria, these thermodynamic parameters could be derived by assuming a steady-state condition.

The amino acid sequences of the alpha and beta chains of hemoglobin from the snake, Liophis miliaris.

The hemoglobin of Liophis miliaris has unusual properties. The hemoglobin is dimeric in the oxy form, and the cooperativity of O2 binding is very low, but both the Bohr effect and cooperativity are greatly enhanced in the presence of ATP (Matsuura, M. S. A., Ogo, S. H., and Focesi, A., Jr. (1987) Comp. Biochem. Physiol. 86A, 683-687). Four unique chains (2 alpha, 2 beta) can be isolated from the hemolysate. The amino acid sequences of one alpha and one beta chain have been determined in an effort to understand the functional properties. Comparison of the sequences with those of the alpha and beta chains of human Hb shows the following. (i) All 7 of the residues in the beta chain normally conserved in globins are identical to those of the human chain: Gly(B6), Phe(CD1), His(E7), Leu(F4), His(F8), Lys(H10), and Tyr(HC2), except that the distal His(E7) has been replaced by Gln in the alpha chain. (ii) All heme contact residues in the beta chain are identical with those in the human chain, but two differences are present in the alpha chain: the distal His(E7) is replaced by Gln and Met(B13) by Leu. (iii) All residues that form the binding site for organic phosphates are identical to those in human Hb. (iv) The major residues that contribute to the normal Bohr effect in human Hb, Asp-beta 94, His-beta 146, and Val-alpha 1 are conserved. (v) All beta chain residues at the alpha 1 beta 2 interface are identical with those in the human chain except two: Glu(G3)----Val and Glu(CD2)----Thr; these differences in charged residues may explain the dissociation to dimers. (vi) The 23 residues of the alpha chain in the alpha 1 beta 2 contact region are identical with those of the human chain except three: Phe(B14)----Leu, Thr(C3)----Gln and Pro(CD2)----Ser. (vii) A total of 17 differences occur at the alpha 1 beta 1 interface, 11 in the alpha chain and 6 in the beta chain.

The amino acid sequences of chains a, b, and c that form the trimer subunit of the extracellular hemoglobin from Lumbricus terrestris.

The extracellular hemoglobin of Lumbricus terrestris comprises four major heme-containing chains, a, b, c, and d in equal proportions. We have determined the amino acid sequences of chains a, b, and c which form a disulfide-linked trimer. Chains a, b, and c have 151, 145, and 153 residues and calculated molecular weights of 17,525, 16,254, and 17,289, respectively. The sequence of chain b, reported previously (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 287, 9005-9015) has been completely redetermined and found to contain 12 fewer residues than originally reported. Chains a and c both contain unusual, highly polar NH2-terminal extensions of 7 residues before the A helix. These segments must be close together because they are joined by a disulfide bond. We suggest that this structure, with seven negatively charged groups, may be part of a functionally important Ca2+-binding site in the trimer. Comparison of the sequences of chains a, b, and c with those of chain d (Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and Walz, D. A. (1987) J. Biol. Chem. 262, 3123-3131) and the four chains of the hemoglobin of Tylorrhynchus heterochaetus (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267) shows that the number and positions of the cysteinyl residues are all conserved. This suggests that the extracellular hemoglobins from both the Oligochaeta and Polychaeta have the same number and configuration of disulfide bonds within the molecule. Phylogenetic analysis suggests that gene duplication first generated an intracellular hemoglobin branch and an extracellular hemoglobin branch. DNA coding for a signal peptide would have been acquired by the extracellular globin gene after this event. At least two further gene duplications are required to account for the present four polypeptide chains.

Dimer-tetramer transition in hemoglobins from Liophis miliaris--I. Effect of organic polyphosphates.

Hemoglobin from the water-snake Liophis miliaris in the stripped form presents high oxygen affinity of about P50 = 1 mmHg and Hill coefficient of about 1.0 at pH from 6.8 to 8.5. In the presence of ATP such values become P50 = 20 mmHg and nH about 2.0, respectively, at low pH from 6.5 to 7.5. When the pH increases an abrupt decrease of both P50 and nH values occurs falling close to those found for the stripped hemoglobin. Gel-filtration in Sephadex G-100 equilibrated with 0.05 M Tris-HCl buffer containing 1 mM EDTA of the stripped hemoglobin show the presence of only one component of mol. wt of about 32,500 dt similar to the dimer of human hemoglobin A. The deoxy form of the dimer previously treated with ATP and placed on Sephadex column in the same condition but containing 1 mM IHP emerges as tetramer with mol. wt similar to that found for human hemoglobin, i.e. of about 65,000 dt. Results of the multiplicity of the snake hemoglobin, as well as the large alkaline Bohr effect in the presence of ATP previously reported, seems to be inconsistent due to the dimer-tetramer transition that occurs when ATP is bound to the stripped hemoglobin. A molecular mechanism involving the dimer-tetramer transition is proposed to described the oxygen transport in these animals.

Further evidence of dimer-tetramer transition in hemoglobin from Liophis miliaris.

Liophis miliaris hemoglobins in the stripped form exhibit a high oxygen affinity, a small alkaline Bohr effect, a pronounced organic polyphosphate effect and a pH-dependent Hill coefficient, close to 2 below pH 7.5 and near 1 at higher pHs. Molecular weight determinations indicate 2 forms, a dimeric form of MW 32000 d. and a tetrameric form of about 64000 d. The deoxyhemoglobin is tetrameric. Ion-exchange chromatography shows two components which may bind to each other being eluted as a single component of MW 32000 d. The oxygen alkaline Bohr effect observed for the stripped hemoglobin may be explained by the transition from the tetramer to the dimer during oxygen addition experiments. The phenomenon appears to be unique among animals. beta-Chain sequencing determinations show that abnormal human hemoglobin with similar properties has a glutamic acid residue substituted at the alpha 1-beta 2 interface by valine in snake hemoglobin.

Further evidence of dimer-tetramer transition in hemoglobin from Liophis miliaris

Liophis miliaris hemoglobins in the stripped form exhibit a high oxigen affinity, a small alkaline Bohr effect, a pronounce organic polyphosphate effect and a pH-dependent Hill coefficient, close to 2 below pH 7.5 and near 1 at higher pHs. Molecular weight determinations indicate 2 forms, a dimeric form of MW 32000 d. and a tetrameric form of about 64000 d. The deoxyhemoglobin is tetrameric. Ion-exchange chromatography shows two components which may bind to each other being eluted as a single component of MW 32000 d. The osygen alkaline Bohr effect observed for the stripped hemoglobin may be explained by the transition from the tetramer to the dimer during oxygen addition experiments. The phenomenon appears to be unique among animals. ß-Chain sequencing determinations show that abnormal human hemoglobin with similar properties has a glutamic acid residue substituted at the alfa1-ß2 interface by valine in snake hemoglobin