Physicochemical aspects of carbohydrate binding to some plant lectins with binding preference forN-acetylgalactosamine and galactose

This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO₂Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics. When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A₄ fromGriffonia simplicifolia is a simple bimolecular association with parametersk ₊ = 9.4 × 10⁴ M^-1 s^-1 andk _-1 = 5.3 s^-1 at 23°C, but binding to the GSAI-B₄ lectin is biphasic. The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk _-1 = 0.24 s^-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is 100 times smaller than for other mono-and disaccharides investigated. The binding mechanism is simple and the derivatisation of Galβ1 → 3GalNAc does not affect the affinity to a considerable degree. The binding preference of tetravalentsoybean agglutinin for MeαGalNAc over MeαGal by a factor of 25 is mainly of enthalpic origin with an additional 7 kJ mol^-1; the NAc group causes perturbation of a tryptophanyl residue, evidenced by protein difference absorption spectrometry. In the glycosides, a large aglycon likeβpNO₂ Phe orβMeUmb hardly affects the affinity of SBA but a largeN-dansyl group increases the affinity by a factor 20 as compared to GalNAc. The 10-fold increase in carbohydrate-specificN-dansylgalactosamine fluorescence, together with a very favourable entropic contribution point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The dissociation-rate parameter of the MeUmbβGalNAc SBA complex is slower than for any reported monosaccharide-lectin complex: 0.4 s^-1. The divalent lectin fromErythrina cristagalli preferentially binds the Galβ1 → 4GlcNAc structure that occurs in manyN-glycoproteins. The combining site was mapped thermodynamically with carbohydrates ranging from mono-to pentasaccharides as derived fromN-glycoproteins. Here, N-dansylgalactosamine was used as a fluorescent indicator ligand in substitution titrations. When Galβ1 → 4GlcNAc was linkedα1 → 2 orα1 → 6 to Man, the binding enthalpy and entropy remained practically constant. Application of stopped flow kinetics and pressure-jump relaxation withN-dansylgalactosamine gave mono-exponential signal changes with a concentration dependence corresponding tok ₊ = 4.8 x 10⁴ M^-1 s^-1 k _- = 0.4 to 0.66 s^-1 and a change in reaction volume of+7ml/mol.     

Elastic properties of bacterial flagellar filaments. II. Determination of the modulus of rigidity

Elongation of a helical bacterial flagellar filament subjected to fluid flow was calculated on the assumption that one end of the filament is firmly attached to a substratum. It was found that the quantity [E(d/2 pi r)2 + 2 mu] could be determined by measuring the elongation at various flow rates, where E is Young's modulus, mu the modulus of rigidity, r the radius of the helix, and d the helical pitch. Experiments were carried out to determine the above quantity for Salmonella flagellar filaments assuming a close-coil form. Because the above quantity is almost equal to 2 mu for a helical form with a large radius/pitch ratio, we were able to determine the modulus of rigidity for this kind of flagellar filament from plots of elongation vs. flow rates. The modulus of rigidity was determined to be about 1 X 10(11) dyn/cm2, i.e., 2 orders of magnitude larger than the previously estimated value.     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

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.     

Alkaline transfer of DNA to plastic membrane

DNA forms a stable complex with Gene Screen Plus membrane at alkaline pH. Based on this, a method of alkaline transfer of DNA from agarose gel to Gene Screen Plus membrane was elaborated. The procedure entails the use of 0.4 M NaOH for both, the DNA denaturation and DNA transfer steps. The alkaline transfer offers a higher hybridization efficiency and simplifies the transfer procedure as compared with the standard method of DNA transfer at neutral pH. In addition, it can be used to remove RNA contamination from the transferred DNA.     

Population genetics theory of concerted evolution and its application to the immunoglobulin V gene tree

Summary The previous simple model for treating concerted evolution of multigene families has been revised to be compatible with various new observations on the immunoglobulin variable region family and other families. In the previous model, gene conversion and unequal crossing-over were considered, and it was assumed that genes are randomly arranged on the chromosome; neither subdivision nor correlation of gene identity and chromosomal distance were considered. Although this model satisfactorily explains the observed amino acid diversity within and between species, it fails to predict the very ancient branching of the mouse immunoglobulin heavy chain V-gene family. By incorporating subdivided structure and genetic correlation with chromosomal distance into the simple model, the data of divergence may be satisfactorily explained, as well as the rate of nucleotide substitution and the amino acid diversity. The rate at which a V-gene is duplicated or deleted by conversion or by unequal crossing-over is estimated by the new model to be on the order of 10^–6 per year. The model may be applicable to other multigene families, such as those coding for silkmoth chorion or mammalian kallikrein.Contribution no. 1560 from the National Institute of Genetics, Mishima, 411 Japan     

Evolution of catalytic proteins

Summary It is believed that all present-day organisms descended from a common cellular ancestor. Such a cell must have evolved from more primitive and simpler precursors, but neither their organization nor the route such evolution took are accessible to the molecular techniques available today. We propose a mechanism, based on functional properties of enzymes and the kinetics of growth, which allows us to reconstruct the general course of early enzyme evolution. A precursor cell containing very few multifunctional enzymes with low catalytic activities is shown to lead inevitably to descendants with a large number of differentiated monofunctional enzymes with high turnover numbers. Mutation and natural selection for faster growth are shown to be the only conditions necessary for such a change to have occurred.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

Genetics of yellow berry in wheat (Triticum aestivum)

Summary The inheritance of yellow berry, a grain disorder in durum and bread wheats, was studied in six intervarietal crosses in bread wheat. The trait was found to be controlled by either two or three dominant genes. Monosomic analysis using Chinese Spring monosomic series showed the presence of two major dominant genes on chromosomes 1A and 7A, and four modifiers on 4A, 4B, 6A and 6D, which influence the expression of yellow berry in bread wheat.