Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds: Study of Their Interaction with Exogenous Proteinases

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.     

Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low K_m) when compared with available published data for other laccase isozymes. The specificity constant (k_cat/K_m) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (k_cat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.     

Inhibition by N′-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in α-ketoglutarate amination

The effect of N′-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the α-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K_i, estimated as 199?μmol?l^?1 at 25°C and pH 8.0.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10^–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10^–6M) to 7.1 ([TPT] = 10^–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7)·10³M^–1.     

Dynamic behaviour of inflorescence-bearing Triticale and Triticum stems

The mechanical response of cereal plant shoots to loads caused by wind and gravity in the field is swaying in flexure around the vertical or near vertical transient equilibrium position determined by the stationary component of the wind pressure. The aim of this work was to characterise the kinematic and dynamic attributes and their interrelations in freely swaying inflorescence-bearing stems of wheat (Triticum aestivum L.) and Triticale. The fundamental natural frequency of the stems appeared to be considerably lower than predicted from the theory of vibration using the model of a cantilever beam oscillator and assuming the spring constant to be equal to the force-deflection ratio. Because of the rate of deformation and visco-elastic behaviour of the plant material, a discrepancy of about 10% was found between the dynamic and static stem bending resistance. The presence of the tip inflorescence caused vibrating vertical stems to behave as compressed columns in which the effective spring constant was strongly biased by the apical load due to the weight of the inflorescence. At the late milk stage, in the freely swaying stems of wheat and Triticale, the resistance to dynamic lateral loads was reduced by about 30% as a result of compression exerted by the inflorescence. So the prominent effect of the tip inflorescence on the dynamic behaviour (the effective spring constant and the natural frequency) of the stem is attributed to the non-negligible magnitude of the inflorescence weight relative to the critical load producing elastic buckling in slender vertical structures. Stem softening as a consequence of increasing inflorescence weight is assumed to be one of the essential factors reducing the lodging resistance in cereal crops at the late milk stage. The feasibility of the compressed-column approach for predicting the dynamic bending performance of slender vertical plant organs is discussed. Received: 4 March 1998 / Accepted: 20 July 1998     

Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms

Actin binding proteins control actin assembly and disassembly by altering the critical concentration and by changing the kinetics of polymerization. All of these control mechanisms in some way or the other make use of the energy of hydrolysis of actin-bound ATP. Capping of barbed filament ends increases the critical concentration as long as ATP hydrolysis maintains a difference in the actin monomer binding constants of the two ends. A further increase in the critical concentration on adding a second cap, tropomodulin, to the other, pointed filament end also requires ATP hydrolysis as described by the model presented here. Changes in the critical concentration are amplified into much larger changes of the monomer pool by actin sequestering proteins, provided their actin binding equilibrium constants fall within a relatively narrow range around the values for the two critical concentrations of actin. Cofilin greatly speeds up treadmilling, which requires ATP hydroysis, by increasing the rate constant of depolymerization. Profilin increases the rate of elongation at the barbed filament end, coupled to a lowering of the critical concentration, only if ATP hydrolysis makes profilin binding to the barbed end independent of its binding constant for actin monomers.     

Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore

Kinetic studies of macromolecular ligand-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1 : 1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a simple bimolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms     

Dynamic force microscopy for imaging of viruses under physiological conditions

Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized. Published: June 29, 2004.     

Inclusion complexation behavior of dyestuff guest molecules by a bridged bis(cyclomaltoheptaose)[bis(beta-cyclodextrin)] with a pyromellitic acid diamide tether

A novel bridged bis(beta-cyclodextrin) with a pyromellitic acid 2,5-diamide tether (2) has been synthesized by reaction of 6(I)-(2-aminoethyleneamino)-6-deoxycyclomaltoheptaose [mono 6-(2-aminoethyleneamino)-6-deoxy-beta-cyclodextrin] with 1,2,4,5-benzenetetracarboxylic dianhydride. Its inclusion complexation behavior with some representative dyestuffs, i.e., Acridine Red (AR), Rhodamine B (RhB), Neutral Red (NR), Brilliant Green (BG), was studied by using UV-absorption, fluorescence, and 2D NMR spectroscopy. Fluorescence titrations have been performed at 25 degrees C in pH 7.2 buffer solution to calculate the binding constants of resulting complexes. These results obtained indicated that bis(beta-cyclodextrin) 2 exhibits the strongly enhanced binding ability with all dye molecules examined compared with natural cyclodextrins. The binding modes of 2 with dye molecules have been deduced by 2D NMR experiments to establish the correlations between molecular conformations and binding constants of inclusion complexation. It is found that the improved binding ability and molecular selectivity of 2 could be attributed to double-cavity cooperative inclusion interaction and the size/shape matching between the host and guest.     

Program MULDER -- a tool for extracting torsion angles from NMR data

MULDER (Mostly UniversaL Dihedral angle ExtractoR) is a program for extraction of torsion angle information from NMR data. Currently, it can analyze two types of input data: The torsion angle data, where several ³J-coupling constants and/or interatomic distances are combined in order to reduce the torsion angle ambiguity arising from solving the isolated Karplus (or distance) equation, and the sugar pucker data, where the dynamics of five-membered sugar rings is evaluated by postprocessing the results calculated from ³J_(HH) coupling constants by program PSEUROT. Program MULDER can be used either as an alternative to r-MD programs in situations where only specific structural features are studied, or as a preparatory tool in connection with full r-MD structure calculation for extraction of unambiguous torsion angle restraints.