Acetylcholinesterase: Role of the enzyme's charge distribution in steering charged ligands toward the active site

The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42 Å radius centered in the enzyme monomer and separated from the protein surface by I-14 Å is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32 Å radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a neutral ligand. However, the differences among the encounter rates for the various mutants considered here are not pronounced, all rate constants being within ±10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzyme's total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzyme's charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it has entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase. © 1996 John Wiley & Sons, Inc.     

AutoMotif server: prediction of single residue post-translational modifications in proteins

The AutoMotif Server allows for identification of post-translational modification (PTM) sites in proteins based only on local sequence information. The local sequence preferences of short segments around PTM residues are described here as linear functional motifs (LFMs). Sequence models for all types of PTMs are trained by support vector machine on short-sequence fragments of proteins in the current release of Swiss-Prot database (phosphorylation by various protein kinases, sulfation, acetylation, methylation, amidation, etc.). The accuracy of the identification is estimated using the standard leave-one-out procedure. The sensitivities for all types of short LFMs are in the range of 70%. AVAILABILITY: The AutoMotif Server is available free for academic use at http://automotif.bioinfo.pl/     

Peptide loading in the endoplasmic reticulum accelerates trafficking of peptide: MHC class II complexes in B cells

In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-A^b and an antigenic peptide E52–68 covalently linked to its -chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, E52–68:I-A^b complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.     

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.     

Effects of bunaftine on morphology,microfilament integrity,and mitotic activity in cultured human fibroblasts and HeLa cells

Summary Human fibroblasts and HeLa cells were treated with bunaftine (N-butyl-N-/2-(diethylamino)ethyl/-1-naphthalenecarboxamide) in vitro. At concentrations of 0.5–2.0 mM, the drug caused contraction and rounding of the cells with loss of microvilli-like processes. Aggregates of dense, partly granular, partly fibrillar material formed in the cytoplasm and the rough endoplasmic reticulum became vesiculated. Immunofiuorescence microscopy with DNase I and anti-DNase I demonstrated that bundles of actin filaments were disrupted, forming rings, coils, and granules. Filaments stained with antibodies to vimentin (fibroblasts) and prekeratin (HeLa cells) showed less characteristic rearrangements, probably related to the rounding up of the cells. 0.4 mM bunaftine increased and 0.8–1.0 mM markedly decreased the percentage of mitotic cells, without accumulation of cells in any particular stage of mitosis. The drug may arrest the cell cycle at some point before mitosis; it may have a critical concentration above which the arrest becomes permanent.These results suggest that bunaftine interferes with the integrity of microfilament bundles in a different manner from that of cytochalasins. It does not cause any depletion of cellular ATP, indicating that its effect is not a result of inhibition of cell metabolism. It is proposed that bunaftine may be used as a complement to cytochalasins in studies of the microfilament system of the cell. The possible binding of bunaftine to actin or myosin and further details of its mechanism of action remain to be elucidated.     

Photoinhibition of white clover seed germination at low water potential

Photosensitivity of germination of white clover ( Trifolium repens L. cv. Podkowa) seeds was studied under water deficit (low water potential) conditions at 25°C. The seeds showed negative photoblastism, which was most pronounced at -0.03 MPa polyethylene glycol solution. Inhibition was observed at two different wavelength bands with maxima at 660 nm (R) and around 730 nm (FR). Red light acted identically to white light (maximum inhibition ca 50%). The effect of far-red illumination was less inhibitory (20–30%). The photoresponse required long illuminations (3 h exposures); saturation level was at 0.1 W m⁻², independently of the light quality. White clover seed germination showed no reversibility of the effects of R and FR light. Prolonged illumination with R and FR increased the inhibition, and intermittent illumination had a higher effect than a continuous one. It was concluded that the photoinhibition of germination of seeds of Trifolium repens involves a reaction dependent on the rate of phytochrome interconversion, a property that is characteristic for the high irradiance reaction.     

Hemodynamic and bioelectric disturbances in striated muscles of rats subjected to accelerative forces after a period of hypokinesia

A series of experimental investigations are described concerning the influence of hypokinesia, acceleration and associated effect of hypokinesia and acceleration in different periods of time on the displacement of plasma proteins and on bioelectric activity of striated muscles. Disturbances in hemodynamic and bioelectric activity of striated muscles by these two factors are discussed.