Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Molecular characterization of human platelet glycoproteins IIIa and IIb and the subunits of the latter

Sedimentation equilibrium and low-angle laser-light scattering were used to determine the molar mass of the glycoprotein moieties in the complexes of sodium dodecyl sulphate with the human platelet membrane glycoproteins IIb (GPIIb), IIIa (GPIIIa), and the (GPIIb) and (GPIIb) subunits of GPIIb. The values obtained by both procedures, except those for GPIIb, agree within experimental error with those calculated from their chemical composition: GPIIb (114,000 g mol^-1), GPIIb (22,200 g mol^-1), and GPIIIa (91,500 g mol^-1). The molar mass of GPIIb determined by light scattering (142,000 g mol^-1) and sedimentation equilibrium at different solvent densities (134,000 g mol^-1) also agree, within experimental error, with the values calculated either from its chemical composition (136,500 g mol^-1) or from the sum of the molar masses of its subunits. However the molar mass determined by sedimentation equilibrium at constant solvent density, is consistently underestimated (116,000 g mol^-1).High-performance size-exclusion chromatography in sodium dodecyl sulphate solutions overestimates the molar mass of these glycoproteins and their Stokes radii, and therefore the maximal frictional ratios derived from them.Abbreviations GPIIb glycoprotein IIb - GPIIIa glycoprotein IIIa - GPIIb and GPIIb and subunits of GPIIb, respectively - CM-GPIIb CM-GPIIb, and CM-GPIIIa, totally reduced and carboxymethylated forms of GPIIb, GPIIb, and GPIIIa, respectively - SDS sodium dodecyl sulphate - eosin-ITC eosin-5-isothiocyanate     

Chromosome aberration and ploidy equilibrium of Vicia faba in tissue culture

Summary Different phytohormone concentrations induced different fequencies of various chromosome aberrations in calli of Vicia faba. NAA 10 ppm plus KT 2.5 ppm produced more haploids and NAA 30 ppm plus NAA 7.5 ppm produced more tetraploids and breakage. The relationship among the aberrations was analyzed. The hypothesis of ploidy equilibrium was established. The chromosome doubling rate and reduction rate of each treated group were calculated in relation to the observed data and the hypothesis. The frequency of tetraploids and breakage are correlated with each other. The frequency of total aberrations is linearly correlated with that of micronucleus formation. The regression equation is x=31.92+ 10.67 y.     

Prospective theoretical and experimental study towards electrochemically grafted poly(N-vinyl-2-pyrrolidone) films on metallic surfaces

Quantum chemistry calculations of equilibrium geometry, atomic charges, dipole moment, and frontier orbital energies are carried out on model N-vinyl-2-pyrrolidone, its radical anion and cation, and three protonated derivatives. Attempts to obtain poly(N-vinyl-2-pyrrolidone) films grafted on nickel and platinum electrodes, respectively by cathodic and anodic polarizations, are reported. A thin, covering and insulating film is obtained on the Pt anode, as indicated by surface characterizations carried out with UPS and IRRAS spectroscopies. The fact that the film remains adherent to the metallic surface in spite of a permanent contact with an acetonitrile solution in which poly(N-vinyl-2-pyrrolidone) is soluble suggests that chemisorption has indeed been achieved.     

How can quantum mechanics of material evolution be possible?: Symmetry and symmetry-breaking in protobiological evolution

Material self-assembly as a self-organizing process is always accompanied by symmetry-breaking in the material configuration. Self-sequencing of amino acids during their thermal polymerization has lost a certain property of permutation symmetry that was observed in the mixture of free amino acids. The evolutionary precursor state is more symmetrical about its internal material configuration and more degenerate due to the multitude of the indistinguishable individuals. The evolution proceeds in the direction along which the degeneracy in the internal states dissolves owing to the symmetry-breaking originating in material flow equilibrium of open material aggregates. Protobiological information is latent in the material system which is highly symmetrical and highly degenerate in its internal states. Evolution of matter is an endogenous process in which the earlier symmetric property is lost and less degenerate states are approached. Quantum-mechanically, the generation of protobiological information is due to the symmetry-breaking of the Hamiltonian originating in the interaction with the exterior through material flow, in contrast to the Schrödinger equation which preserves a symmetry and the associated invariants.     

Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

Comparison of serum free thyroxine measurements by chemiluminescence and equilibrium dialysis

A study of 239 patients compared free thyroxine (FT4) measurements made by equilibrium dialysis (ED) with measurements made using the Magic Lite FT4 chemiluminescence (Cl) immunoassay (Ciba Corning Immunodiagnostics). Patient groups: 41 normals; 27 hyperthyroid; 29 hypothyroid; 37 sick euthyroid; 10 chronic renal failure (CRF) and 25 pregnant patients; 13 oestrogen; 10 heparin; 12 salicylate; and 9 dilantin-treated patients; 3 lipaemic; 5 haemolysed; 6 hyperbilirubinaemic patients; 6 low thyroid binding protein (TBP) and 6 high TBP level patients. The two assays gave comparable results in most groups. Both assays tended to give elevated values in heparinized patients but FT4–ED results were more obviously affected. Pregnant patients and women on oral oestrogen had higher mean values with FT4–ED. In both assays the sick euthyroid and CRF patients had mean FT4 values similar to healthy euthyroid patients; the range of values in sick euthyroid and CRF patients was similar in both assays but wider than in healthy euthyroid patients. A supplemental study of 81 unselected acutely ill patients using FT4–Cl alone confirmed the wider range of values to be anticipated in sick euthyroid patients.     

Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions

Column chromatography of the Escherichia coli mannitol permease (mannitol-specific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)-dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho-exchange between mannitol and mannitol 1-phosphate (a partial reaction in the overall PEP-dependent phosphorylation of mannitol). Kinetic analysis of the phospho-exchange reaction revealed that Pi stimulates phospho-exchange by increasing the Vmax of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less-stable conformation of the dimer) is hypothesized to be involved in the initial mannitol-binding and PEP-dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1-phosphate.     

Compositional statistics: An improvement of evolutionary parsimony and its application to deep branches in the tree of life

Summary We present compositional statistics, a new method of phylogenetic inference, which is an extension of evolutionary parsimony. Compositional statistics takes account of the base composition of the compared sequences by using nucleotide positions that evolutionary parsimony ignores. It shares with evolutionary parsimony the features of rate invariance and the fundamental distinction between transitions and transversions. Of the presently available methods of phylogenetic inference, compositional statistics is based on the fewest and mildest assumptions about the mode of DNA sequence evolution. It is therefore applicable to phylogenetic studies of the most distantly related organisms or molecules. This was illustrated by analyzing conservative positions in the DNA sequences of the large subunit of RNA polymerase from three archaebacterial groups, a eubacterium, a chloroplast, and the three eukaryotic polymerases. Internally consistent results, which are in accord with our knowledge of organelle origin and archaebacterial physiology, were achieved.     

Prediction of the Reaction Equilibrium of Biocatalytic Reactions in Aqueous-Organic Two-Phase Systems

Biocatalytic reactions can be carried out in aqueous-organic two-phase systems. Several models to describe the thermodynamically-determined equilibrium position in such systems have appeared in the literature. Some of these models are only valid for dilute systems, whereas others can also be used for nondilute systems. In this paper, these models are described and compared. It is explained in what way the equilibrium constants of each model can be used to predict the product concentration in different organic solvents.