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1.
Mol Biol (Mosk) ; 54(3): 474-479, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32492011

RESUMO

The iron-containing protein neuroglobin (Ngb) involved in the transport of oxygen is generally considered the precursor of all animal globins. In this report, we studied the structure of Ngb of the cold-water sponge Halisarca dujardinii. In sponges, the oldest multicellular organisms, the Ngb gene contains three introns. In contrast to human Ngb, its promoter contains a TATA-box, rather than CG-rich motifs. In sponges, Ngb consists of 169 amino acids showing rather low similarity with its mammalian orthologues. It lacks Glu and Arg residues in positions required for prevention of hypoxia-related apoptosis. Nevertheless, Ngb contains both proximal and distal conserved heme-biding histidines. The primary structure of H. dujardinii neuroglobin predicted by sequencing was confirmed by mass-spectrometry analysis of recombinant Ngb expressed in E. coli. The high level of Ngb expression in sponge tissues suggests its possible involvement in the gas metabolism and presumably in other key metabolic processes in H. dujardinii.


Assuntos
Neuroglobina/química , Poríferos/química , Aminoácidos , Animais , Escherichia coli , Íntrons , Regiões Promotoras Genéticas
3.
Bioorg Khim ; 37(1): 45-54, 2011.
Artigo em Russo | MEDLINE | ID: mdl-21460880

RESUMO

Proteolytic degradation of autoantigens is of prime importance in current biochemistry and immunology. The most fundamental issue in this field is the functional role of peptides produced when the specificity of hydrolysis changes during the shift from health to disease and from normal state to pathology. The identification of specific peptide fragments in many cases proposes the diagnostic and prognostic criterion in the pathology progression. The aim of this work is comparative study of the degradation peculiarities of one of the main neuroantigen, myelin basic protein by proteases, activated during progress of pathological demyelinating process, and by proteasome of different origin. The comparison of specificity of different studied biocatalysts gives reason to discuss the critical change in the set of myelin basic protein fragments capable to be presented by major histocompatibility complex class I during neurodegeneration, which can promote the progress of autoimmune pathological process.


Assuntos
Proteína Básica da Mielina/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Animais , Calpaína/metabolismo , Catepsina D/metabolismo , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Proteômica , Tripsina/metabolismo
5.
Acta Naturae ; 1(1): 84-7, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22649589

RESUMO

The proteasome is a high molecular protein complex whose purpose is specific protein degradation in eukaryotic cells. One of the proteasome functions is to produce peptides, which will then be presented on the outer cell membrane using main histocompatibility complex (MHC) molecules of the first or second class. There are definite reasons to believe that proteasome directly takes part in the specific degradation of myelin basic protein (MBP), which make up to 30% of all proteins in the myelin sheath of neuronal axons. The details of the proteasomal degradation of MBP are still unclear. In this work, the features of specific MBP degradation by proteasome were studied.It was demonstrated that MBP (non-ubiquitinated) is a good substrate for 20S and for the 26S proteasome. This is the first work on detecting the sites of MBP proteolysis by proteasome from brains of SJL/J/J and Balb/C mice's lines. Substantial differences in the degradation pattern of this neuroantigen were found, which could indicate the better presentation MBP parts on MHC molecules in the case of mice predisposed to the development of experimental autoimmune encephalomyelitis.

6.
Bioorg Khim ; 34(3): 376-81, 2008.
Artigo em Russo | MEDLINE | ID: mdl-18672688

RESUMO

A chemoenzymatic syntheses was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are i-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for substrate (I). The lowest detectable papain concentrations were 2.4 x 10(-10) M for (I) and 1.2 x 10(-11) M for (II). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.


Assuntos
Compostos de Anilina/síntese química , Quimotripsina/química , Cumarínicos/síntese química , Corantes Fluorescentes/síntese química , Papaína/química , ortoaminobenzoatos/síntese química , Compostos de Anilina/química , Catálise , Cumarínicos/química , Dipeptídeos/síntese química , Dipeptídeos/química , Corantes Fluorescentes/química , Especificidade por Substrato , ortoaminobenzoatos/química
7.
Prikl Biokhim Mikrobiol ; 44(3): 270-5, 2008.
Artigo em Russo | MEDLINE | ID: mdl-18663948

RESUMO

Commercial preparations of trypsin, varying in activity, were immobilized in a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In an organic solvent environment, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-argininine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.


Assuntos
Enzimas Imobilizadas/química , Oligopeptídeos/síntese química , Álcool de Polivinil/química , Tripsina/química , Animais , Estabilidade Enzimática , Géis/química , Suínos
8.
Bioorg Khim ; 31(6): 586-92, 2005.
Artigo em Russo | MEDLINE | ID: mdl-16363130

RESUMO

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.


Assuntos
Enzimas Imobilizadas/química , Peptídeos/síntese química , Álcool de Polivinil/química , Subtilisinas/química , Aminoácidos/química , Bacillus subtilis/enzimologia , Géis , Estereoisomerismo
9.
Biochemistry (Mosc) ; 68(11): 1261-6, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14640970

RESUMO

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Peptídeos/química , Subtilisinas/química , Resinas Acrílicas , Estabilidade Enzimática , Cinética , Álcool de Polivinil , Subtilisinas/isolamento & purificação
10.
Bioorg Khim ; 29(5): 551-8, 2003.
Artigo em Russo | MEDLINE | ID: mdl-14601410

RESUMO

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.


Assuntos
Peptídeos/síntese química , Subtilisinas/metabolismo , Água/química , Catálise
11.
Bioorg Khim ; 27(5): 347-51, 2001.
Artigo em Russo | MEDLINE | ID: mdl-11641908

RESUMO

The segment condensation of peptides on a solid phase (Aminosilochrom) in organic medium catalyzed by a subtilisin complex with sodium dodecylsulfate was studied. The dependence of the efficiency of the enzymatic coupling of tripeptides with the basic structure X-Ala-Ala-Y-OMe [where X = Z, Boc, or Dnp and Y = Leu or Glu(OMe)] on the spacer content on the support and on the structure of the acylating component was investigated. The tripeptide segments were successively coupled to Aminosilochrom containing the Met-Ala-Gly spacer, and the peptidylaminosilochroms Dnp-Ala-Ala-Leu-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Dnp-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A (A is the Aminosilochrom residue) were obtained in satisfactory yields. It was shown by these examples that the second and third segments are attached in yields higher than that for the first segment and the coupling efficiency does not depend on the amino acid composition of the acylating component.


Assuntos
Peptídeos/síntese química , Dióxido de Silício , Aminoácidos , Peptídeos/química
12.
Bioorg Med Chem Lett ; 11(8): 1005-8, 2001 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-11327576

RESUMO

Serine proteinase subtilisin 72 was covalently attached to the beads of poly(vinyl alcohol)-cryogel, a macroporous hydrogel prepared by the freeze-thaw technique. The immobilized enzyme was examined as a catalyst in the synthesis of protected peptides Z-Ala-Ala-Xaa-Phe-pNA (Xaa = Leu, Glu, Lys) in acetonitrile/dimethylformamide mixtures. Immobilized subtilisin catalyzed with high yield the formation of peptide bonds between Phe-pNA and acyl donors including those with free carboxylic group and non-protected C-terminal basic and acidic amino acid residues.


Assuntos
Acetonitrilas/farmacologia , Dimetilformamida/farmacologia , Peptídeos/síntese química , Subtilisinas/efeitos dos fármacos , Subtilisinas/metabolismo , Acetonitrilas/química , Bacillus subtilis/enzimologia , Catálise , Dimetilformamida/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/efeitos dos fármacos , Enzimas Imobilizadas/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Biossíntese Peptídica , Peptídeos/química , Peptídeos/metabolismo , Álcool de Polivinil/química , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Fatores de Tempo
13.
Bioorg Khim ; 26(6): 411-6, 2000 Jun.
Artigo em Russo | MEDLINE | ID: mdl-10923188

RESUMO

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using the Met residue cleavage by BrCN.


Assuntos
Peptídeos/química , Dodecilsulfato de Sódio/química , Subtilisinas/química
14.
Bioorg Khim ; 25(8): 591-6, 1999 Aug.
Artigo em Russo | MEDLINE | ID: mdl-10578464

RESUMO

The solubility, stability, and activity of native subtilisin 72 and of its complex with SDS were comparatively studied in a number of polar organic solvents. Subtilisin was found to catalyze peptide bond formation when suspended in acetonitrile or solubilized as a complex with SDS in ethanol and isopropanol. Tripeptide Z-Ala-Ala-Leu-pNA, tetrapeptides A-Ala-Ala-P1-P1'-B, where A = Z or Abz; P1 = Leu, Phe, Met, Trp, Ile, Tyr, Phe(NO2), or Glu(OMe), P1' = Leu, Phe, Glu, Ala, Ile, Val, or Arg; B = NH2, pNA, or 2-(2,4-dinitrophenyl)aminoethylamine residue (Ded); pentapeptides Z-Ala-Ala-Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA; and hexapeptide Abz-Val-Ala-Phe-Phe-Ala-Ala-Ded were synthesized using the SDS-subtilisin complex. The complex also efficiently catalyzed the oligomerization of tripeptide H-Phe-Ala-Leu-OCH3 in ethanol, which resulted in a 63:37 mixture of trioligomer and tetraoligomer. It was demonstrated that SDS-subtilisin is a much more efficient catalyst than the suspension of native enzyme.


Assuntos
Peptídeos/síntese química , Subtilisinas/metabolismo , Catálise , Estabilidade Enzimática , Solventes
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