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1.
Biochemistry (Mosc) ; 66(4): 390-6, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11403645

RESUMO

Binding of 1,8-anilinonaphthalene sulfonate (1,8-ANS) with native human oxyhemoglobin (Hb) in 50 mM potassium phosphate buffer (pH 7.4) was studied by steady-state fluorescence spectroscopy and by laser spectrofluorimetry with subnanosecond time resolution. The distribution of fluorescence decay times and parameters of two- and three-exponential deconvolution of the fluorescence kinetics of 1,8-ANS in Hb solution demonstrate that the emission at wavelengths lambdaem of 455-600 nm is not single-exponential and has components with mean decay times < 0.5, 3.1-5.5, and 12.4-15.1 nsec with the amplitudes depending on the emission wavelength. Analysis of time-resolved fluorescence spectra shows that the shortest-lived component should be assigned to 1,8-ANS molecules in the aqueous medium, whereas the two longer-lived components are assigned to two types of binding sites for 1,8-ANS in the Hb molecule characterized by different polarity and accessibility to water molecules.


Assuntos
Naftalenossulfonato de Anilina/química , Naftalenossulfonato de Anilina/metabolismo , Corantes Fluorescentes/análise , Oxiemoglobinas/química , Oxiemoglobinas/metabolismo , Naftalenossulfonato de Anilina/análise , Sítios de Ligação/fisiologia , Fluorescência , Corantes Fluorescentes/química , Humanos , Cinética , Oxiemoglobinas/análise , Espectrometria de Fluorescência/métodos
2.
J Photochem Photobiol B ; 58(2-3): 156-62, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11233644

RESUMO

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.


Assuntos
Naftalenossulfonato de Anilina/metabolismo , Corantes Fluorescentes/metabolismo , Oxiemoglobinas/metabolismo , Sítios de Ligação , Feminino , Humanos , Técnicas In Vitro , Oxiemoglobinas/química , Oxiemoglobinas/efeitos da radiação , Fotoquímica , Soluções , Espectrometria de Fluorescência
3.
Prikl Biokhim Mikrobiol ; 31(3): 308-10, 1995.
Artigo em Russo | MEDLINE | ID: mdl-7638156

RESUMO

The use of barium salts of ribose 1-phosphate and 2-deoxyribose-1-phosphate as the donors of the carbohydrate part of the required nucleosides in the reaction of enzymatic glycosylation of heterocyclic bases was studied. It was shown that application of indicated substrates in the synthesis of modified nucleosides-ribothymidine and bromovinyldeoxyuridine, catalyzed by the nucleoside phosphorylases from Escherichia coli BM-11, leads to the 96-98% yield of required products even at an equimolar ratio of original pentose 1-phosphates and heterocyclic bases in the reaction mixtures.


Assuntos
Compostos de Bário/química , Bromodesoxiuridina/análogos & derivados , Pentosefosfatos/química , Pentosiltransferases/metabolismo , Uridina/análogos & derivados , Bromodesoxiuridina/metabolismo , Catálise , Escherichia coli/enzimologia , Fermentação , Glicosilação , Indicadores e Reagentes , Uridina/biossíntese
4.
Appl Microbiol Biotechnol ; 32(6): 658-61, 1990 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1367439

RESUMO

Synthesis of 9-(beta-D-arabinofuranosyl)guanine (ara-G) from 1-(beta-D-arabinofuranosyl)cytosine (ara-C) and guanine, guanosine or 2'-deoxyguanosine (dG) by glutaraldehyde-treated Escherichia coli BM-11 cells is described. It is shown that the concentration of phosphate ions, molar ratio of substrates and pH of the reaction medium are factors affecting product yield. Under optimum conditions ara-G was produced in the reaction mixture in a yield of 63%-65% based on dG as the best source of guanine base. The yield of isolated ara-G was 48%-53%.


Assuntos
Arabinonucleosídeos/biossíntese , Escherichia coli/metabolismo , Arabinonucleosídeos/isolamento & purificação , Proteínas de Bactérias/metabolismo , Citarabina/metabolismo , Citosina Desaminase , Glutaral , Guanina/metabolismo , Nucleotídeos de Guanina/metabolismo , Nucleosídeo Desaminases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Uridina Fosforilase/metabolismo
5.
Biokhimiia ; 49(4): 570-6, 1984 Apr.
Artigo em Russo | MEDLINE | ID: mdl-6733160

RESUMO

The interaction of the 8-aza-16-oxasteroid series (8-AS) with cytochrome P-450 from liver microsomes of intact and phenobarbital-induced rats has been studied. It has been shown that 8-AS are the substrates for the cytochrome P-450-dependent enzyme system, and that their affinity for cytochrome P-450 is determined by the structure of the compounds tested. Using inhibitory analysis, the site in the active center of hemoprotein responsible for 8-AS binding was examined. The possibility of direct participation of the 8-AS ketogroup in their binding with cytochrome P-450 is discussed.


Assuntos
Azasteroides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Esteroides Heterocíclicos/metabolismo , Animais , Sítios de Ligação , Colesterol/farmacologia , Técnicas In Vitro , Masculino , Metirapona/farmacologia , Fenobarbital/farmacologia , Propranolol/farmacologia , Ratos , Ratos Endogâmicos , Espectrofotometria , Relação Estrutura-Atividade
6.
FEBS Lett ; 164(2): 247-50, 1983 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-6653790

RESUMO

The differential scanning microcalorimetry and fluorescence methods, using probes ANS and pyrene, have been employed to study thermotropic behaviour of rat liver microsomes in the presence and absence of Mg2+. Addition of Mg2+ yields three partially reversible phase transitions at 18, 27 and 32 degrees C, respectively. A character of Mg2+-induced rearrangements in a membrane and their relation to a catalytic function of a cytochrome P-450-dependent enzymatic system is discussed.


Assuntos
Magnésio/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Temperatura , Animais , Varredura Diferencial de Calorimetria , Ratos , Espectrometria de Fluorescência
7.
Biochim Biophys Acta ; 692(2): 287-95, 1982 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-7171597

RESUMO

The interaction of rat liver microsomal cytochrome P-450 with phospholipids has been investigated employing differential scanning microcalorimetry. It is shown that the thermotropic behaviour of phospholipid vesicles reconstituted with cytochrome P-450 depends on liposome composition, protein concentration and the mode of reconstituted system preparation. From the delta H dependence on protein concentration in proteoliposomes it was calculated that one cytochrome P-450 molecule influence 350 +/- 50 dimyristoylphosphatidylcholine (DMPC) molecules. The electrostatic interaction of cytochrome P-450 and negatively charged phospholipid, phosphatidylinositol (PI), mixed with DMPC involves the temperature stabilization of proteoliposomes at a phase transition of phospholipid bilayers. The thermal denaturation temperature is increased due to negatively charged PI added.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Lipossomos , Microssomos Hepáticos/metabolismo , Fosfolipídeos/farmacologia , Animais , Varredura Diferencial de Calorimetria , Dimiristoilfosfatidilcolina , Cinética , Masculino , Fosfatidilcolinas , Fosfatidilinositóis , Proteolipídeos , Ratos , Ratos Endogâmicos , Temperatura
8.
Biokhimiia ; 42(12): 2110-5, 1977 Dec.
Artigo em Russo | MEDLINE | ID: mdl-597513

RESUMO

The kinetics of aniline hydroxylation with: 1) rat liver microsomes involving NADPH and O2 (System I); 2) hepatic microsomes and tertiary butylhydroperoxide (System II) and 3) microsomes and cumyl hydroperoxide (System III) within 15--37 degrees C has been studied. The reactions were characterized by the values of the aniline oxidation rate constants, k2=v/[E]0, where [E]0 is the initial concentration of cytochrome P--450: k1 2=1,60.10(8) exp (--13400/RT) sec-1., k2 2=1,66.10(9) exp (--14500/RT) sec-1., k3 2=6,83.10(9) exp (--15300/RT) sec-1. The values of delta H* and delta S* were calculated and compared for these three systems. A conclusion is drawn that the act of oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned system.


Assuntos
Anilina Hidroxilase/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Cinética , Ratos , Termodinâmica
9.
Chem Biol Interact ; 18(2): 195-204, 1977 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19154

RESUMO

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15--37 degrees C. The reactions were characterized by the values of the aniline oxidation rate constants, k2 = V/E0, where E0 is the initial concentration of cytochrome P-450: K 1/2 = 1.60 - 10(8) EXP (-13 400/RT) sec-1, k 2/2 = 1.66 - 10(9) exp (-14 500/RT) sec-1, k 3/2 = 6.83 - 10(9) exp (-15 300/RT) sec-1. The values of delta H0 and delta S0, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.


Assuntos
Compostos de Anilina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Anilina Hidroxilase/metabolismo , Animais , Derivados de Benzeno , Compostos de Benzil/farmacologia , Feminino , Cinética , NADP/farmacologia , Consumo de Oxigênio , Peróxidos/farmacologia , Ratos , Temperatura , Termodinâmica
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