PubMed Health⌕ Search

Biomedical subjects

A Arnone

Publications and source records attributed to A Arnone.

15 recordsLinked to original sources
PubMed1992

High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from beta-globins having mutated amino termini.

The crystal structures of three mutant hemoglobins reconstituted from recombinant beta chains and authentic human alpha chains have been determined in the deoxy state at 1.8-A resolution. The primary structures of the mutant hemoglobins differ at the beta-chain amino terminus. One mutant, beta Met, is characterized by the addition of a methionine at the amino terminus. The other two hemoglobins are characterized by substitution of Val 1 beta with either a methionine, beta V1M, or an alanine, beta V1A. All the mutation-induced structural perturbations are small intrasubunit changes that are localized to the immediate vicinity of the beta-chain amino terminus. In the beta Met and beta V1A mutants, the mobility of the beta-chain amino terminus increases and the electron density of an associated inorganic anion is decreased. In contrast, the beta-chain amino terminus of the beta V1M mutant becomes less mobile, and the inorganic anion binds with increased affinity. These structural differences can be correlated with functional data for the mutant hemoglobins [Doyle, M. L., Lew, G., DeYoung, A., Kwiatkowski, L., Noble, R. W., & Ackers, G. K. (1992) Biochemistry preceding paper is this issue] as well as with the properties of ruminant hemoglobins and a mechanism [Perutz, M., & Imai, K. (1980) J. Mol. Biol. 136, 183-191] that relates the intrasubunit interactions of the beta-chain amino terminus to changes in oxygen affinity. Since the structures of the mutant deoxyhemoglobins show only subtle differences from the structure of deoxyhemoglobin A, it is concluded that any of the three hemoglobins could probably function as a surrogate for hemoglobin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1992

A third quaternary structure of human hemoglobin A at 1.7-A resolution.

Previous crystallographic studies have shown that human hemoglobin A can adopt two stable quaternary structures, one for deoxyhemoglobin (the T-state) and one for liganded hemoglobin (the R-state). In this paper we report our finding of a second quaternary structure (the R2-state) for liganded hemoglobin A. The magnitudes of the spatial differences between the R- and R2-states are as large as those between the R- and T-states. Of particular interest are the structural changes that occur as a result of R-T and R-R2 transitions at the so-called "switch" region of the critical alpha 1 beta 2 interface. In the R-state, His-97 beta 2 is positioned between Thr-38 alpha 1 and Thr-41 alpha 1, whereas in transition to the T-state His 97 beta 2 must "jump" a turn in the alpha 1 C helix to form nonpolar contacts with Thr-41 alpha 1 and Pro-44 alpha 1. This facet of the R-T transition presents a major steric barrier to the quaternary structure change. In the R2-state, His-97 beta 2 simply rotates away from threonines 38 alpha 1 and 41 alpha 1, breaking contact with these residues and allowing water access to the center of the alpha 1 beta 2 interface. With the switch region in an open position in the R2-state, His-97 beta 2 should be able to move by Thr-41 alpha 1 and make the transition to the T-state with a steric barrier that is less than that for the R-T transition. Thus the R2-state may function as a stable intermediate along a R-R2-T pathway. The T-, R-, and R2-states must coexist in solution. That is, the fact that these states can be crystallized implies that they are all energetically accessible structures. What remains to be determined are the T-to-R, T-to-R2, and R-to-R2 equilibrium constants for hemoglobin under various solution conditions and ligation states. Although this may prove to be difficult, we discuss previously published results which indicate that low concentrations of inorganic anions or low pH may favor the R2-state and at least one alpha 1 beta 2 interface mutation stabilizes a quaternary structure that is very similar to the R2-state.

Amino Acid Sequence↗
PubMed1992

Synthesis and pharmacological evaluation of enantiomerically pure 4-deoxy-4-fluoromuscarines.

Four isomers of [(4-fluoro-5-methyl-tetrahydrofuran-2-yl)methyl]trimethylammonium iodide (4-deoxy-4-fluoro-muscarines) were prepared in enantiomerically and diastereomerically pure form from (S)-(-)-methyl 4-methylphenyl sulfoxide, ethyl fluoroacetate, and allyl bromide. Their absolute configurations were assigned by 1H NMR analyses. The four optically pure compounds were tested in vitro on guinea pig and their muscarinic potency was evaluated at M3 (ileum and bladder) and M2 (heart) muscarinic receptor subtypes. Compound 1a, the most potent isomer of the series, was also tested in vivo on pithed rat and its muscarinic activity at the M1 receptor subtype was compared with that of muscarine. Moreover, affinity and relative efficacy were calculated in vitro for this compound at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The 4-deoxy-4-fluoromuscarines display a similar trend of potency as the corresponding muscarines and compound 1a shows differences in the affinity constants among the studied tissues. Replacement of a hydroxyl group for a fluorine atom in the 4 position of muscarine produces 1 order of magnitude increase in affinity for cardiac M2 muscarinic receptors controlling rate, while the affinity at cardiac M2 muscarinic receptors controlling force is unchanged, opening the possibility of a further classification of cardiac muscarinic receptors.

Animals↗
PubMed1992

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.

Anions↗
PubMed1978

Crystalline enzyme.substrate complexes of asparate aminotransferase.

Crystalline complexes of cytoplasmic aspartate aminotransferase of pig heart with the substrates L-glutamate and L-aspartate, and with other amino acids, have been prepared and polarized light absorption spectra have been measured. Striking differences in the directions of polarization of the absorption bands are seen. A complete half-transamination of pyridoxal phosphate to pyridoxamine phosphate by aspartate or by cysteine sulfinate can be demonstrated in the crystal as can the accumulation of a quinonoid intermediate with erythro-beta-hydroxyaspartate. X-ray diffraction studies show that the crystals with erythro-beta-hydroxyaspartate and alpha-methylaspartate are isomorphous with those of both alpha and beta subforms of the native enzyme.

Amino Acids↗
PubMed1977

Specific modification of the alpha chain C-terminal carboxyl group of hemoglobin by trypsin-catalyzed hydrazinolysis.

In human deoxyhemoglobin a salt bridge links the alpha carboxyl of Arg-141 of each alpha chain to the epsilon-amino group of Lys-127 of the opposite alpha chain. These salt bridges are believed to contribute to the constraints in the quaternary deoxy (T) structure that lower its oxygen affinity. We have tested this hypothesis by incubating hemoglobin with 2 M hydrazine and trypsin which catalyzes specifically the reversible hydrazinolysis of the alpha carboxyl of Arg-141alpha. X-ray analysis shows the major structural difference between native deoxyhemoglobin and hydrazide deoxyhemoglobin to be the loss of the Arg-141alpha1-Lys-127alpha2 salt bridge and its Arg-141alpha2-Lys-127alpha1 counterpart. Accurate oxygen equilibrium curves of hydrazide hemoglobin show that blocking of the salt bridge has raised the oxygen affinity of the T structure while leaving that of the quaternary oxy (R) structure unchanged.

Amino Acid Sequence↗
PubMed1977

The binding of zinc to human deoxyhemoglobin and its possible relevance to the anti-sickling effect of zinc.

We find only one major zinc binding site in crystals of deoxyhemoglobin A. It is located at an interface between adjacent tetramers where residues histidine 116, histidine 117, and glutamate 26 on the beta 1 chain of one tetramer are in close proximity to lysine 16 and glutamate 116 on the alpha 2 chain of a neighboring tetramer. If this intertetramer contact does not exist in the rigid fibers of polymerized deoxyhemoglobin S, then low levels of zinc may promote its formation at random points along the fiber and thereby inhibit the formation of long fibers.

Anemia, Sickle Cell↗
PubMed1976

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.

Allosteric Site↗