Inhibition of myosin ATPase by metal fluoride complexes. (1/348)

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.  (+info)

Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching. (2/348)

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.  (+info)

Resolution and characterization of tryptophyl fluorescence of hen egg-white lysozyme by quenching- and time-resolved spectroscopy. (3/348)

The fluorescence spectral distributions of four tryptophan residues of hen egg-white lysozyme were analyzed using time-resolved and quenching-resolved fluorescence spectroscopy. Trp62 and Trp108 gave the fluorescence maxima at 352 nm and 342 nm, respectively. The fluorescence of Trp28 and Trp111 occurred only at 300-360 nm and they were observed as an unresolved emission band with a maximum and shoulder at 320 nm and 330 nm. The fluorescence quenching and decay parameters of each tryptophan residue reconfirmed that Trp62 was fully exposed to the solvent but Trp108 was sealed in the cage of the peptide chains and furthermore showed that Trp28 and Trp111 are under the influence of the larger fluctuational motion at the hydrophobic matrix box. The fluorescence responses of each tryptophan residue to the lysozyme-ligand interaction suggested that the internal fluctuation was reduced by the binding of ligand to give a distorted conformation to the hydrophobic matrix box region.  (+info)

Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif. (4/348)

The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5' RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources.  (+info)

Changes in thyroid gland morphology after acute acrylamide exposure. (5/348)

High exposure to the acrylamide monomer has been associated with neuropathy and neurotoxic effects. Chronic lower exposure causes endocrine disruption associated with thyroid, testicular, and mammary tumors. To investigate mechanisms of endocrine disruption, short-term, low-level oral dosing studies were conducted. Weanling female Fischer 344 rats were acclimatized for two weeks before dosing. Controls were given distilled water by gavage and rats in other groups were given acrylamide at doses of 2 mg/kg/day and 15 mg/kg/day for 2 or 7 days by gavage. Twenty-four h after the last dose, the rats were killed by decapitation. Trunk blood was collected for hormone analyses and tissues for histopathological examination. There were no toxicity-related deaths, no clinical signs of toxicity, and no significant difference in the mean body weight of animal groups. Histopathological examination of select tissues showed no lesions of pathologic significance. Plasma thyroxine (T4), thyroid stimulating hormone (TSH), prolactin (PRL), and pituitary TSH and PRL analyses did not reveal significant changes between control vs. treated rats. In the 7-day study, however, there was a slight dose-dependent increase in plasma T4 and a slight dose-dependent decrease in plasma TSH. Thyroid gland morphometry showed a significant (p < 0.05) decrease in the colloid area and a significant increase (p < 0.05) in the follicular cell height of treated rats as compared to controls. The follicular area shrinkage was similar in both studies. These results show a very early endocrine response to very low levels of toxic insult and opens other venues to further investigate the mechanisms of endocrine disruption by acrylamide.  (+info)

Detection of aneuploidy by multicolor FISH in mouse sperm after in vivo treatment with acrylamide, colchicine, diazepam or thiabendazole. (6/348)

Multicolor fluorescence in situ hybridization (FISH) was used to investigate the induction of aneuploidy during meiosis in young adult male mice treated with chemicals chosen for the EU sponsored aneuploidy project (acrylamide, colchicine, diazepam and thiabendazole). The aim of the present study was to evaluate the frequency of aneuploid sperm induced by each of these chemicals by sperm FISH. Male (102/ElxC3H/El)F1 mice were treated with acrylamide (120 and 60 mg/kg single dose i.p.), colchicine (1.5 and 3 mg/kg single dose, i.p.), diazepam (300, 150 and 75 mg/kg single dose by oral intubation) or thiabendazole (100 and 300 mg/kg daily for 11 days by oral intubation). At 22 days after the last treatment, sperm were collected from the cauda epididymis. Three chromosome FISH was applied to determine hyperhaploid and diploid sperm with DNA probes specific for the chromosomes X, Y and 8. Five animals were treated per dose group and sperm aneuploidy was evaluated in 10,000 sperm per animal. We found significant increases in the frequency of total hyperhaploidy for the males treated with 3.0 mg/kg colchicine (0.092 versus 0.056%, P < 0.05) and with 1.5 mg/kg colchicine (0.082 versus 0.050%, P < 0.05), as well for the males treated with 300 mg/kg diazepam (0.081 versus 0.050%, P < 0.05), indicating that colchicine and diazepam each induced germ cell aneuploidy. We also found significant increases in the frequency of total diploidy for the males treated with 300 mg/kg diazepam (P < 0.05) and with 300 mg/kg thiabendazole (P < 0.05). No significant effects were found for 120 and 60 mg/kg acrylamide or for the other doses of diazepam and thiabendazole. These first results indicate that the multicolor FISH method is useful to determine aneuploidy induction in sperm of mice.  (+info)

Effect of acrylamide on aldolase structure. I. Induction of intermediate states. (7/348)

Acrylamide is a fluorescence quencher frequently applied for analysis of protein fluorophores exposure with the silent assumption that it does not affect the native structure of protein. In this report, it is shown that quenching of tryptophan residues in aldolase is a time-dependent process. The Stern-Volmer constant increases from 1.32 to 2.01 M-1 during the first 100 s of incubation of aldolase with acrylamide. Two tryptophan residues/subunit are accessible to quenching after 100 s of aldolase interaction with acrylamide. Up to about 1.2 M acrylamide concentration enzyme inactivation is reversible. Independent analyses of the changes of enzyme activity, 1ANS fluorescence during its displacement from aldolase active-site, UV-difference spectra and near-UV CD spectra were carried out to monitor the transition of aldolase structure. From these measurements a stepwise transformation of aldolase molecules from native state (N) through intermediates: I1, T, I2, to denatured (D) state is concluded. The maxima of I1, T, I2 and D states populations occur at 0.2, 1.0, 2.0 and above 3.0 M of acrylamide concentration, respectively. Above 3.5 M, acrylamide aldolase molecules become irreversibly inactivated.  (+info)

Effect of acrylamide on aldolase structure. II. Characterization of aldolase unfolding intermediates. (8/348)

Molecules of muscle aldolase A exposed to acrylamide change their conformation via I1, T, I2, D intermediates [1] and undergo a slow irreversible chemical modification of thiol groups. There is no direct correlation between activity loss and thiol groups modification. In the native enzyme two classes of Trp residues of 1. 8 ns and 4.9 ns fluorescence lifetime have been found. Acrylamide (0. 2-0.5 M) increases lifetime of longer-lived component, yet the transfer of aldolase molecules even from higher (1.0 M) perturbant concentration to a buffer, allows regain original Trp fluorescence lifetime. I1, detected at about 0.2 M acrylamide, represents low populated tetramers of preserved enzyme activity. T, of maximum population at about 0.7-1.0 M acrylamide, consists of meta-stable tetramers of partial enzymatic activity. These molecules are able to exchange their subunits with aldolase C in opposition to the native molecules. At transition point for I2 appearance (1.8 M acrylamide), aldolase becomes highly unstable: part of molecules dissociate into subunits which in the absence of perturbant are able to reassociate into active tetramers, the remaining part undergoes irreversible denaturation and aggregation. Some expansion of aldolase tetramers takes place prior to dissociation. D, observed above 3.0 M acrylamide, consists of irreversibly denatured enzyme molecules.  (+info)