Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Spatial structure of the basic chain of the neurotoxin I molecule from the venom of cobra Naja naja oxiana and its crystal packing

The structure of the alpha-carbon chain was solved by molecular replacement method at 2.7 A resolution. Neurotoxin I (NTX-I) is one of the main protein components purified from the venom of the central asian cobra Naja naja oxiana. NTX-I is known to bind specifically to acetylcholine receptors thus preventing the transmission of the neuroconductivity signal from synapses to muscles. NTX-I crystals were grown either by vapour diffusion or dialysis methods using specially prepared microdialysis cell. The intensities of reflections from native NTX-I crystals were measured in the range of 38.0-2.1 A-1 by omega-scan method with a Syntex P21 diffractometer operated in automatic regime. To determine the position and mode of packing of NTX-I molecule in unit cell program packages MERLOT and BLANC were applied running on a NORD-500 computer.     

Magnetic interaction of manganese with pheophytin anion-radical and chlorophyll cation-radical in reaction centers of the photosystem II

The influence of Mn on saturation curves of ESR spectra of Ph(-) and P(+)(680) at 1-200K in samples with different content of Mn has been studied. An analysis of these data and those on photoinduced changes of fluorescence yield of chlorophyll leads to the conclusion that the Mn-containing centre in Photosystem 2 is a cluster of 4 Mn atoms, two of which can be replaced by Mg(2+) or any other divalent metal. The distances between Mn Na Ph as well as between Mn and P(680) have been estimated.     

Manometric determination of biological degradability of substances poorly soluble in aqueous environments

Several ways or methods of testing the biological degradability of organic substances poorly soluble in aqueous environments were verified by applying manometric BOD measurements. Model substrates employed here were dibutyl phthalate, dioctyl adipate, bio-fuel and some of their components. Dosages of substrates were varied by combining direct weighing and sonification, by using an emulsion with Triton X100, or by a solution in 1,1,2-trichlorotrifluoroethane. These selected variants of dosage, except for the last, proved to be equivalent from the viewpoint of degree and rate of biological degradation. The applied methods gave values of biochemical/theoretical oxygen demand ratio (BOD/ThOD) around 50%, even for substrates claimed to be biologically easily degradable.     

Mass spectral characterization of two onion constituents with prostaglandin E-like activity as lipoxygenase metabolites of linoleic acid

Onions ($\text{Allium cepa}$ have been used in folk medicine for the treatment of hypertension and gastrointestinal ulcers, which are cases where the administration of prostaglandins is considered. Using bioassay guided fractionation and gas chromatography/mass spectrometry (GC/MS) we have isolated two biologically active fractions with PGE-like properties and have characterized these products as lipoxygenase metabolites of linoleic acid.For the isolation of the active principles, onion bulbs were homogenized in phosphate buffer and the PGE-like products were isolated using Amberlite XAD-2 absorption, silicic acid column chromatography and silicagel thin layer chromatography. The PGE-like activity was estimated in a cascade superfusion system in which the isolated rabbit coeliac artery, the rabbit mesenteric artery and the rat fundus were used as assay organs.For the GC/MS characterization, various types of volatile derivatives were prepared in order to facilitate the structure elucidation. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation. The active fractions yielded identical electron-impact mass spectra, indicating that they are stereoisomeric, and each fraction was identified as a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic and of 9,10,13-trihydroxy-11-octadecenoic acid. With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butyl boronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol function.The isomeric trihydroxylated octadecenoic acids have been described for the first time as metabolites of linoleic acid in wheat flour incubates. In this system, the trihydroxylated octadecenoic acids were shown to be formed through a sequence of three reactions, including an initial 9- or 13-lipoxygenation yielding hydroperoxy octadecadienoic acids, followed by rearrangement into unstable allylic epoxy hydroxy octadecenoic acids, which in turn are hydrolyzed to trihydroxy octadecenoic acids. Furthermore, structurally related trihydroxy octadecadienoic acids have also recently been isolated from a plant source, more specifically from the roots of $\text{Bryonia Alba}$, which are used for the same medicinal purposes as onion bulbs.     

Dynamic structure of metallothionein

Molecular sieve chromatography of rabbit liver metallothionein at different electrolyte concentrations revealed that this protein undergoes an increase in Stokes radius from 1.50 to 1.78 nm when the ionic strength is lowered from 0.5 to 0.015 indicating a change in molecular shape and/or hydration. The variation in ionic strength also affects the far-UV circular dichroism of metallothionein reflecting a conformational transition in the protein. The effects are attributed to changes in intramolecular repulsion between the strongly negatively charged metal-thiolate clusters of the protein. It is suggested that metallothionein exists in at least two interchangeable conformational states which differ in hydrodynamic properties and whose equilibrium concentrations are determined by the electrostatic free energy of the system.