Excited chlorophyll and related problems.

A historical outline is presented of the primary light energy conversion in photosynthesis studied by our research group. We found that photoexcited chlorophylls, pheophytins and porphyrins are capable of reversible and irreversible oxido-reduction. The mechanism of the photosensitized electron transfer from donor to acceptor molecule is based on the reversible photochemical oxido-reduction of the pigment-sensitizer.

Photogeneration of NADH under coupled action of CdS semiconductor and hydrogenase from Alcaligenes eutrophus without exogenous mediators.

Photoreduction of NAD has been accomplished by a system consisting of the NAD-dependent hydrogenase from Alcaligenes eutrophus immobilized on CdS particles with formate as artificial electron donor. Enzymatically active NADH is formed under illumination of this system by visible light. Accumulation of the coenzyme dimer (NAD)2 was not detected.

Reversed micelles of polymeric surfactants in nonpolar organic solvents. A new microheterogeneous medium for enzymatic reactions.

A new microheterogeneous non-aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n-butanol mixtures. The hydrodynamic radius of polymeric-reversed micelles was estimated to be in the range 22-51 nm, depending on the water content of the system, as determined by means of the quasi-elastic laser-light scattering.

pH dependence of fluorescence and absorbance spectra of free sulphonated aluminium phthalocyanine and its conjugate with monoclonal antibodies.

Water-soluble phthalocyanines and phthalocyanines linked to targeting monoclonal antibodies are considered to be one of the most promising photosensitizers in photodynamic therapy. Here the spectrum characteristics of sulphonated aluminium phthalocyanine and its protein conjugate in the pH range 1.8-12.

GTPase activity of bacteriophage T4 sheath protein.

We show by nuclear magnetic resonance studies that, following GTP hydrolysis during phage T4 sheath contraction, GDP remains bound to the sheath protein (gp18), whereas orthophosphate is released. gp18 in the contracted state has GTPase activity and can hydrolyse exogenous GTP; the reaction is calcium-dependent and displays high substrate specificity. The process comprises two steps: (1) displacement of GDP from gp18 by exogenous GTP, and (2) GTP hydrolysis proper.

Laccase-based biosensor for determination of polyphenols: determination of catechols in tea.

A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized laccase. Analytical systems of flow injection and batch types were considered.

A new approach to the construction of potentiometric immunosensors.

An electrochemical immunosensor based on a new detection principle was developed. Laccase, which is able to catalyse the electroreduction of oxygen via the direct (mediatorless) mechanism was used as an enzyme label. The new detection method does not require the presence of an electrochemically active mediator, and the reaction substrates are atmospheric oxygen and electrons, the latter being taken by the active site of the enzyme label directly from the electrode.

The main adenosine triphosphate-binding component of amphibian oocytes is ferritin.

After fractionation of mitochondrion-free extracts of Xenopus laevis and Rana temporaria oocytes in sucrose gradients, a distinct peak of adenosine triphosphate (ATP)/guanosine triphosphate (GTP)-binding activity in the 50-70 S range has been detected. This substance has a boyant density in Cs2SO4 of 1.45 g/cm3.

Expression and regulation of casein kinase 2 during heat shock in Verticillium dahliae.

A homogeneous preparation of casein kinase 2 has been isolated from the phytopathogenic fungus Verticillium dahliae (the parasite of cotton). The enzyme consists of three subunits with molecular masses of 53, 41, and 38 kDa. Highly specific immune serum against casein kinase 2 has been obtained.

The effect of water content and nature of organic solvent on enzyme activity in low-water media. A quantitative description.

A simple theoretical model was suggested to describe quantitatively the effect of water content and nature of organic solvents on catalytic behavior of enzymes suspended in low-water media. The model was based on a generally accepted notion that the destruction of the protein hydration shell is one of the main reasons for protein denaturation by organic solvents. The validity of the model was confirmed by the example of catalytic behavior of immobilized laccase suspended in water/organic mixtures of different compositions.

Purification and properties of a high-molecular-mass complex between Val-tRNA synthetase and the heavy form of elongation factor 1 from mammalian cells.

In extracts of various mammalian tissues obtained in the presence of protease inhibitors Val-tRNA synthetase exists exclusively as a complex with a molecular mass of about 800 kDa. This complex was purified by gel filtration and two HPLC steps and contained five different polypeptides with molecular masses of 140, 50, 50, 40 and 30 kDa. The complex seems to have no tissue or species specificity, because preparations with identical polypeptide composition were obtained by the same method from rabbit liver and reticulocytes, and rat and beef liver.

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves : Assignment of spectral bands.

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627.

Relationship between surface hydrophilicity of a protein and its stability against denaturation by organic solvents.

The stability of alpha-chymotrypsin covalently modified with a strongly hydrophilic modifier, pyromellitic dianhydride, against solvent-induced denaturation in water-organic solvent binary mixtures has been studied. It was found that the hydrophilization results in a strong stabilization of the enzyme against denaturation by organic solvents. The stabilizing effect is explained in terms of the enhanced ability of the hydrophilized enzyme to keep its hydration shell, which is indispensable for supporting the native protein conformation, from denaturing stripping by organic solvents.

Evidence for non-uniform translation of individual polypeptides in rat liver mitochondria.

[35S]Methionine labelled mitochondrial translation products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 8 M urea. Following fluorography and scanning, the synthesis time of major polypeptides was determined. It was found that: (1) from 9 to 16 min are required to synthesize various complete polypeptides whose size differs by a factor of more than 5.

pH-dependent changes in structure and RNA-binding activity of casein kinase 2 from Rana temporaria oocytes.

It is demonstrated by filter-binding assay that casein kinase 2 from Rana temporaria oocytes binds rRNA in vitro with high affinity. Ligand-blotting shows that rRNA-binding activity is inherent to alpha and alpha' subunits of the enzyme. Increase of pH from 6.

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e.

Flow-injection glucose determination with long-wavelength luminescent oxygen probes.

A flow-injection method for the determination of glucose in serum is presented. It is based on the enzymatic measurement of oxygen consumption detected via oxygen quenching of the luminescence of certain metalloporphyrins. Phosphorescent water-soluble Pt2+ and Pd(2+)-porphyrins have been characterized by luminescence spectroscopy and decay-time measurements in various buffers, and found to be suitable for oxygen detection in biological systems.

The effect of alkali light chains on the thermal stability of myosin subfragment 1.

Thermal denaturation of myosin subfragment 1 (S1) isoforms from rabbit skeletal muscle containing the different alkali light chains A1 and A2 [S1(A1) and S1(A2), respectively] were studied by various methods. Turbidity measurements showed that thermally induced (heating rate 1 degrees C min-1) aggregation of S1(A1) occurs at lower temperatures than that of S1(A2). However, the temperature dependences of the tryptophan fluorescence spectrum and that for ATPase inactivation were the same for S1(A1) and S1(A2).

Adaptation of an HPLC system for transient-state enzyme kinetic experiments: pulse-flow method.

The components of an HPLC system can be easily reassembled into an instrument for transient-state enzyme kinetic experiments based on the continuous-flow technique. The scheme of reassembly, operation and data evaluation is described in detail for the Pharmacia FPLC system. The working quality of the suggested scheme was tested using a well-studied process of bacterial beta-galactosidase activation by Mg2+ ions.

Photodestruction in vitro of tumour cells sensitized by porphyrins and their conjugates with specific antibodies.

The photodynamic action of a number of carboxylic porphyrins and their covalent conjugates with specific monoclonal IgM antibodies on a human lung adenocarcinoma cell line was studied. All the conjugates showed strong phototoxicity which did not correspond with the phototoxicity of the free porphyrins. Absolute quantum yields of singlet oxygen photogeneration by the porphyrins and their conjugates were obtained and compared with the phototoxicity of the compounds.

Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase.

A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test.

Studies on receptor interaction of ceruloplasmin with human red blood cells.

The interaction of CP with the red blood cell (RBC) receptor was shown to be a Ca2+ dependent process and be limited by CP binding on RBC membrane which is not followed by CP transport through the membrane into RBC. The nature of receptor interaction was determined. It was shown that receptors are formed by glycoproteins of PAS1 and PAS2 (glycoforin dimer and monomer, respectively) and terminal residues of sialic acid of these glycoproteins are important for CP reception.

Structure and properties of a Cephalosporium acremonium alpha-galactosidase.

The amino acid and sugar composition of the enzyme protein, the effect of urea, sodium dodecyl sulphate and Concanavalin A on the purified alpha-galactosidase (EC 3.2.1.

Quantitative determination of phospholipids using the dyes Victoria blue R and B.

Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm. Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes. This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube.