Quenching of the fluorescence of Tyr and Trp residues of firefly luciferase from Luciola mingrelica by the substrates.

Luciferase of the firefly Luciola mingrelica is characterized by fluorescence of not only the unique Trp residue (lambda(em) = 340 nm), but also that of Tyr residues (lambda(em) = 308 nm). Quenching of the intrinsic fluorescence of the luciferase by its substrates luciferin and ATP (AMP) has been studied. Luciferin (LH2) quenches Trp fluorescence more efficiently than the fluorescence of Tyr residues. Two centers of quenching of Tyr fluorescence by ATP have been found corresponding apparently to the allosteric and active sites of the luciferase with K(s(ATP)) = 20 and 110 microM, respectively. The influence of one substrate on the affinity of luciferase to the second was investigated using fluorescence. ATP (AMP) binding to the allosteric sites of the luciferase significantly affects the affinity of luciferase to LH2. Formation of the complex between the luciferase and LH2 affects the affinity of both allosteric and active sites of the luciferase to ATP (AMP). The observed effects are probably connected with conformational changes in the luciferase molecule upon its interaction with the substrates.

Interaction of dimethyl- and monomethyloxyluciferin with recombinant wild-type and mutant firefly luciferases.

Dissociation constants (Ks) in the pH range 6.5-9.0 for complexes of luciferin, dimethyloxyluciferin (DMOL), and monomethylluciferin (MMOL) with recombinant wild-type and mutant (His433Tyr) luciferases from the Luciola mingrelica firefly were determined by fluorescent titration. The protonated effectors were bound by the wild-type and mutant luciferases better than the nonprotonated ones. The affinity of DMOL for the mutant luciferase was higher than for the wild-type luciferase at alkaline pH, whereas the affinity of MMOL was higher at all pH values studied. The fluorescence emission and excitation spectra of DMOL and MMOL in buffer solution (pH 7.8) were obtained in the absence and presence of luciferase. The fluorescence maxima of DMOL and MMOL complexes with luciferase were 20 and 100 nm, respectively, shifted to shorter wavelengths as compared to the values in buffer solution. This was explained by nonspecific and specific influence of the protein microenvironment on the fluorescence spectra of DMOL and its specific influence on the MMOL fluorescence spectra.

Dimethyl- and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase.

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.

[Elements of yoga therapy in the combined rehabilitation of myocardial infarct patients in the functional recovery period]. / Elementy iogoterapii v komplesknoi reabilitatsii bol'nykh infarktom miokarda v funktsional'no-vosstanovitel'nom periode.

Fifty-nine postmyocardial infarction patients received combined therapy involving chemotherapy, physiotherapy, therapeutic exercises and yoga therapy. Thirty-seven controls received the same treatment without yoga exercise. The yoga complex implied elementary simple positions, relaxation exercise and respiratory exercise. A clinical response evident in both the groups appeared more pronounced in the test group as shown by marked improvement in external respiration and blood counts, in exercise tolerance and psychosomatic condition of the patients.