ENDOGENOUS CHANGES OF PHOTOCHEMICAL ACTIVITIES OF SPINACH LEAVES

The activities of green cell-free extracts of spinach leavesin performing photochemical transphosphorylation, photosyntheticphosphorylation, the HILL reaction and the light-induced formationof the endogenous reducing substance (ascorbic acid) were followedin parallel during the growth process of the plant. There wasa certain parallelism between the development of the activitiesof photochemical transphosphorylation, of photo-synthetic phosphorylationand of the HILL reaction, activities being low in the earlierstage of growth, reaching a maximum just before efflorescence,and showing thereafter a more or less sharp decline. The activityin the light-induced formation of endogenous reducing substancewas undetectable for the first 35 day-period of growth, reacheda maximum about one week earlier than the other three activities,and again disappeared after 60 days of growth. (Received September 9, 1960; )     

The place of inactivated actin and its kinetic predecessor in actin folding-unfolding

The kinetics of actin unfolding induced by guanidine hydrochloride of different concentrations was studied. The parametric representation of the kinetic dependencies of tryptophan fluorescence intensity changes recorded at two wavelengths allowed us to detect and characterize a new essentially unfolded kinetic intermediate. Its characteristics suggested that this intermediate state is a premolten globule. It was shown that the equilibrium transition between inactivated and completely unfolded states is also a two-step process and proceeds via an essentially unfolded kinetic intermediate. The new kinetic pathway of actin unfolding--refolding was proposed. According to it, the founded essentially unfolded kinetic state is the on-pathway intermediate, while inactivated actin is the off-pathway misfolded state stabilized by aggregation of partially folded macromolecules of protein.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Further studies on the role of cell-wall-degrading enzymes in cell-wall loosening in oat coleoptiles

A fungal endo-ß-l,3-glucanase was compared with afungal exo-ß-1,3-glucanase with respect to their effectson elongation and cell-wall extensibility in oat coleoptilesegments. The exo-enzyme enhanced elongation and extensibilityof the cell wall. Its effect was not additive to the effectof indole-3-acetic acid when given together with the latter,at least during 3 hr of incubation. Endo-glucanase showed nosignificant effect on elongation and no interaction with theexo-enzyme. Auxin and exo-glucanase increased extensibilityof the cell wall. The exo-glucanase was separated by isoelectricfocusing. The two fractions which were separated and showedglucanase activity induced elongation and cell wall loosening. (Received March 16, 1970; )     

A water-soluble precursor for efficient silica polymerization by silicateins

Silicateins, the spicule-forming proteins from marine demosponges capable to polymerize silica, are popular objects of biomineralization studies due to their ability to form particles varied in shape and composition under physiological conditions. Despite the occurrence of the many approaches to nanomaterial synthesis using silicateins, biochemical properties of this protein family are poorly characterized. The main reason for this is that tetraethyl orthosilicate (TEOS), the commonly used silica acid precursor, is almost insoluble in water and thus is poorly available for the protein. To solve this problem, we synthesized new water-soluble silica precursor, tetra(glycerol)orthosilicate (TGS), and characterized biochemical properties of the silicatein A1 from marine sponge Latrunculia oparinae. Compared to TEOS, TGS ensured much greater activity of silicatein and was less toxic for the mammalian cell culture. We evaluated optimum conditions for the enzyme - pH range, temperature and TGS concentration. We concluded that TGS is a useful silica acid precursor that can be used for silica particles synthesis and in vivo applications.     

Spectral properties of thioflavin T in solvents with different dielectric properties and in a fibril-incorporated form

The increase in the solvent polarity induces a significant shift of the long-wavelength absorption band of the thioflavin T (ThT) to the shorter wavelengths. This is due to the fact that the positive charge of the ThT molecule (Z = +1e) is unequally and very differently distributed between the benzthiazole and aminobenzene rings in the ground and excited states. Therefore, ThT ground state is stabilized by the orientational interactions of the polar solvent dipoles with the positively charged ThT fragments, whereas the configuration of the solvation shell of the ThT molecule in the excited Franck-Condon state is likely far from being equilibrium. ThT absorption spectrum has the shortest (412 nm) and the longest (450 nm) wavelengths in water and in water being incorporated to the amyloid fibrils, respectively. Intriguingly, the position of the ThT fluorescence spectrum depends on the polarity of solvent to a significantly lesser degree than its absorption spectrum: being excited at 440 nm, ThT has emission with maxima at 493 and 478 nm in water and fibrils, respectively. This can be due to the fact that, in the excited state, the rotational oscillations of the ThT fragments relative to each other prevent establishing equilibrium with the solvent and fluorescence occurs from the partially equilibrium excited stated to the partially equilibrium ground state. For the fibril-incorporated ThT, the maximum of the fluorescence excitation spectrum coincides with the maximum of the long wavelength absorption band (450 nm), whereas for ThT in aqueous and alcohol solutions, additional short-wavelength bands of fluorescence and fluorescence excitation spectra were described (Naiki et al. Anal. Biochem. 1989, 177, 244-249; Le Vine Methods Enzymol. 1999, 309, 274-284). These bands could result either from some fluorescent admixtures (including free benzthiazole and aminobenzene) or from the specific ThT conformers in which benzthiazole and aminobenzene rings, being oriented at phi angle close to 90 or 270 degrees, serve as independent chromophores. On the basis of the results of the quantum-chemical calculations, it is proposed that at phi = 90 degrees (270 degrees), the relatively low barrier (only 700 cm-1) of the internal rotation of the benzthiazole and aminobenzene rings relative to each other gives rise to a subpopulation of ThT molecules possessing a violated system of the pi-conjugated bonds of the benzthiazole and aminobenzene rings.     

Evolutionary history of tall fescue morphotypes inferred from molecular phylogenetics of the <Emphasis Type="Italic">Lolium</Emphasis>-<Emphasis Type="Italic">Festuca</Emphasis>species complex

Background  

The agriculturally important pasture grass tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum (Schreb.) Darbysh.) is an outbreeding allohexaploid, that may be more accurately described as a species complex consisting of three major (Continental, Mediterranean and rhizomatous) morphotypes. Observation of hybrid infertility in some crossing combinations between morphotypes suggests the possibility of independent origins from different diploid progenitors. This study aims to clarify the evolutionary relationships between each tall fescue morphotype through phylogenetic analysis using two low-copy nuclear genes (encoding plastid acetyl-CoA carboxylase [Acc1] and centroradialis [CEN]), the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) and the chloroplast DNA (cpDNA) genome-located matK gene. Other taxa within the closely related Lolium-Festuca species complex were also included in the study, to increase understanding of evolutionary processes in a taxonomic group characterised by multiple inter-specific hybridisation events.     

Comparison of homoeolocus organisation in paired BAC clones from white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and microcolinearity with model legume species

Background  

White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised.     

Expression of recombinant actin 5C from Drosophila in the methylotrophic yeast Pichia pastoris

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.