Three-dimensional structure prediction of the NAD binding site of proton-pumping transhydrogenase from Escherichia coli

A three-dimensional structure of the NAD site of Escerichia coli transhydrogenase has been predicted. The model is based on analysis of conserved residues among the transhydrogenases from five different sources, homologies with enzymes using NAD as cofactors or substrates, hydrophilicity profiles, and secondary structure predictions. The present model supports the hypothesis that there is one binding site, located relatively close to the N-terminus of the α-subunit. The proposed structure spans residues α145 to α287, and it includes five β-strands and five α-helices oriented in a typical open twisted α/β conformation. The amino acid sequence following the GXGXXG dinucleotide binding consensus sequence (residues α172 to α177) correlates exactly to a typical fingerprint region for ADP binding βαβ folds in dinucleotide binding enzymes. In the model, aspartic acid α195 forms hydrogen bonds to one or both hydroxyl groups on the adenosine ribose sugar moiety. Threonine α196 and alanine α256, located at the end of βB and βD, respectively, create a hydrophobic sandwich with the adenine part of NAD buried inside. The nicotinamide part is located in a hydrophobic cleft between αA and βE. Mutagenesis work has been carried out in order to test the predicted model and to determine whether residues within this domain are important for proton pumping directly. All data support the predicted structure, and no residue crucial for proton pumping Was detected. Since no three-dimensional structure of transhydrogenase has been solved, a well based tertiary structure prediction is of great value for further experimental design in trying to elucidate the mechanism of the energy-linked proton pump. © 1995 Wiley-Liss, Inc.     

Synthesis and platelet aggregation inhibition activity of a series of enantiomeric bicyclo[3.2.0]heptane-6-oximinoacetic acids (1)

Opening of racemic epoxide (3) with (3S)- or (3R)-dimethyl-3-(dimethyl-t-butylsilyloxy)oct-1-ynyl aluminum gave two regioisomers, which were separated chromatographically. The separated regioisomers, themselves mixtures of chromatographically inseparable diastereoisomers, were converted into their dicobalthexacarbonyl complexes, which were easily resolved and isolated by chromatography. The individual diastereoisomers were deprotected to give bicyclo[3.2.0]heptan-3-ones, whose absolute stereochemistry was assigned using circular dichroism. One of these compounds, (1R,2R,3S,5R,3'S)-3-(3'-hydroxyoct-1'-ynyl)-bicyclo[3.2.0]++ +heptan-2-ol-6- oximinoacetic acid (11a) was 4.5 times more potent than PGE1 in inhibiting the ADP-induced aggregation of human platelets. The next most potent compound in this series was the "ent-15-epi" compound (11b), which was 0.034 times the potency of PGE1 in the platelet aggregation assay.     

A chlorinated phenylpyrrole antibiotic fromActinoplanes

Two chlorophenyl-containing antibiotics have been isolated from a strain ofActinoplanes (ATCC 33002). Antibiotic A 15104 Y is a chlorinated phenylpyrrole compound whose structure has been confirmed by chemical synthesis. Antibiotic A 15104 Z is a chlorophenol derivative for which a structural formula is proposed on the basis of its physicochemical properties. A 15104 Y shows antimicrobial activity against Gram-positive, Gram-negative, and acid-fast bacteria, yeasts, fungi, and protozoa, while A 15104 Z possesses a low activity against Gram-positive bacteria andTrichophyton. A 15104 Y has a weak activity in curing experimentally infected mice, at a dose that is one-fifth the LD₅₀ for the same species. This is the first example of a chloropyrrole derivative isolated from an actinomycete.     

Chronostratigraphie de la fin de la derniere glaciation,a la lumiere des resultats de l'etude lithostratigraphique et palynologique du site de Maisieres-Canal (Belgique)

The upper part of the palaeoclimatic sequence of Maisières-Canal shows a succession of four mild episodes at thetransition between the Pleniglacial and the Late-Glacial. Those four mild fluctuations are respectively correlated with Langerie, Lascaux, Angles-sur-l'Anglin and Pré-Bölling oscillations.     

The visual pigment and visual cycle of the lobster,Homarus

Summary The visual pigment of the American lobster,Homarus americanus, has been studied in individual isolated rhabdoms by microspectrophotometry. Lobster rhodopsin has _max at 515 nm and is converted by light to a stable metarhodopsin with _max at 490 nm. These figures are in good agreement with corresponding values obtained by Wald and Hubbard (1957) in digitonin extracts. Photoregeneration of rhodopsin to metarhodopsin is also observed. The absorbance spectrum of lobster metarhodopsin is invariant with pH in the range 5.4–9, indicating that even after isomerization of the chromophore fromcis totrans, the binding site of the chromophore remains sequestered from the solvent environment. Total axial density of the lobster rhabdom to unpolarized light is about 0.7.As described for several other Crustacea, aldehyde fixation renders the metarhodopsin susceptible to photobleaching, a process that is faster at alkaline than at neutral or acid pH. Small amounts of a photoproduct with _max at 370 nm are occasionally seen. A slower dark bleaching of lobster rhabdoms (_1/2–2 h) also occurs, frequently through intermediates with absorption similar to metarhodopsin.The molar extinction coefficient of metarhodopsin is about 1.2 times greater than that of rhodopsin, each measured at their respective _max. Isomerization of the chromophore fromcis totrans is accompanied by a change in the orientation of the absorption vector of about 3°. The absorption vector of metarhodopsin is either tilted more steeply into the membrane or is less tightly oriented with respect to the microvillar axes.When living lobsters are kept at room temperature, light adaptation does not result in an accumulation of metarhodopsin. At 4 °C, however, the same adapting lights cause a reduction of rhodopsin and an increase in metarhodopsin. There is thus a temperature-sensitive regeneration mechanism that supplements photoregeneration. Following 1 ms, 0.1 joule xenon flashes that convert about 70% of the rhodopsin to metarhodopsin in vivo, dark regeneration occurs in the living eye with half-times of about 25 and 55 min at 22 °C and 15 °C respectively.This work was supported by USPHS research grant EY 00222 to Yale University. S.N.B. was aided by NIH Postdoctoral Fellowship EY 52378.     

Evaluation by electron microscopy of the integrity of iodinated plasmalipoproteins

Summary Iodination of proteins and lipoproteins is a widely used in vitro labelling procedure in metabolic, autoradiographic and various other studies. However, all available iodination techniques have involved the possible damage to the proteins by self-irradiation, oxidizing agents, the alkaline milieu or by the introduction of iodine into the molecular structure itself. To evaluate the integrity of iodinated lipoproteins, we observed the electron microscopic appearance of normal and iodinated rabbit very low density lipoproteins (VLDL) by negative staining with phosphotungstic acid. Iodination up to a molar iodine/protein ratio of 2.89 did not result in any change of shape, size or aggregating tendency of the particles. No stacks or disk-like particles like those of various hyperlipoproteinemic states were found. We conclude that electron microscopy is a valuable tool in assessing the morphological appearance of lipoprotein iodination, but it should be complemented by other techniques.     

Further studies on bilitranslocase,a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, α and β, of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is α₂-β.     

Changes in cell nuclei during S phase: progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34.

Using multiparameter flow cytometry we have measured the nuclear DNA content of exponentially growing HL-60 cells in conjunction with protein content, nuclear forward light scatter, DNA in situ sensitivity to denaturation, DNA accessibility to 7-aminoactinomycin D (7-AMD), and content of the proliferation-associated proteins: cyclin (PCNA), p105, p34, and Ki-67. Multivariate analysis of the data made it possible to correlate changes in each parameter with the degree of cell advancement through S phase (amount of replicated DNA). A decrease of the protein/DNA ratio, lowered DNA accessibility to 7-AMD, increased sensitivity of DNA to denaturation, and increased ability of isolated nuclei to scatter light all paralleled cell progression through S phase. These changes indicate that during S phase chromatin progressively condenses and suggest that the condensation is associated with the efflux of nonhistone proteins from the nucleus. The increase in the content of the antigen detected by the Ki-67 antibody was observed to exceed the increase in DNA content during S phase and the rate of the Ki-67/DNA increase was higher during the second half of S phase. Thus, this protein appears to be primarily synthesized during S, especially late in S phase, and is degraded in G1. The ratio of cyclin (PCNA)/DNA remained rather constant whereas the contents of p105 and p34 proteins, when expressed per unit of DNA, both decreased during S phase. The data indicate that significant changes in structure and composition of chromatin take place during S phase and suggest that the composition of chromatin associated with the nonreplicated DNA is different compared to chromatin associated with the newly replicated DNA.     

On coenzyme Q orientation in membranes: A linear dichroism study of ubiquinones in a model bilayer

Summary A general approach is developed to interpret linear dichroism (LD) spectra of ubiquinones (Q _n) in host bilayers. Information is reported in terms of guest-host mutual orientation and localization. The overall orientational anisotropy of guest ubiquinone molecules is described by a basic set of limiting orientation/localization modes. Assignments of the UV transitions of the ubiquinone chromophore were obtained by the liquid crystal-linear dichroism technique and molecular orbital (CNDO/S) calculations. The LD spectra of Q _n in the bilayers provided by the lyotropic nematic mesophase exhibited by water solutions of potassium laurate and decanol were interpreted on the basis of the above assignments. The resulting experimental evidence showed a multisite distribution in the host bilayer for the aromatic heads of all the investigated Q _n derivatives except Q₀. The orientational distribution suggested by the LD spectra fits the solubilization model recently proposed by G. Lenaz [J. Membrane Biol. (1988) 104:193–209] for ubiquinone in lipid membranes. Within this model Q _n molecules are located in the midplane and their headgroups oscillate transversally across the membrane. Q ₀ instead has a single site location, close to the polar bilayer interface. Experimental evidence that the headgroup carbonyls tend to grasp the polar interface of the host bilayer was also obtained. Orientation and location distributions of Q _n guest molecules are therefore likely to result from the tendency of their aromatic heads to grasp the polar heads of the host bilayer and from the concurrent tendency of their chains to settle into the hydrocarbon host interior.abbreviations AA average absorption - OD, OD optical densities for plane polarized radiations parallel () and perpendicular () to the sample optical axis - OD OD — OD - EPR electron paramagnetic resonance - LC-LD liquid crystal-linear dichroism - LD linear dichroism - LD _r reduced linear dichroism. - MO molecular orbital - N nematic - NMR nuclear magnetic resonance - S _jj order parameters of the directions j of the transition moments of the guest chromophore - S _ii order parameters of the orientational axes i of the guest molecule with respect to the magnetic field - S _ii order parameters of the axes i of the guest molecules with respect to the bilayer axis a - S _a order parameters of the host bilayer axis a with respect to the orienting magnetic field - _j,i deflection angles between the directions j and the axes i - O _i optical factors of the i axis see Eq. (A4)] - Q_n ubiquinone whose isoprenoid chain contains n isoprenoid units Dr. A. Rossi is gratefully acknowledged for the t.e.m. reduction of the spectra. Ubiquinone homologs were kind gifts from Eisai Co., Tokyo, Japan. This work was supported by M.U.R.S.T., and C.N.R. Target Project on Biotechnology and Bioinstrumentation, Rome, Italy.