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Biomedical subjects

E A Dukhanina

Publications and source records attributed to E A Dukhanina.

At least 19 recordsLinked to original sources
PubMed2001

Molecular mechanisms of different sensitivity of tumor cells to dexamethasone.

The response of two cell lines, CSML-0 (does not express metastasin) and CSML-100 (high expression of metastasin) to cytolytic action of glucocorticoid was studied. Dexamethasone (1 microM) induced apoptosis of CSML-0 cells, while CSLM-100 cells were resistant to its cytolytic action. Apoptotic death of CSLM-100 cells was induced by incubation with dexamethasone in the presence of Ca-ATPase inhibitors, vanadate or thapsigargin. Metastasin, a proteins of the S-100 family, activated Ca-ATPase and ATP-dependent Ca2+ transport in plasmolemmal fraction of CSML-100 cells. Experiments showed that metastasin-induced activation of Ca-ATPase is a possible mechanisms of CSML-100 cell resistance to cytolytic dexamethasone action.

Adenosine Triphosphate↗
PubMed2000

Nonlymphoid cultured cells possess a system controlling cellular compatibility.

We show that various nonlymphoid cultured cells can activate the production of cytotoxic factors in response to direct contact with cells of a different kind. Accumulation of cytotoxic factors in the medium was detected 1 h after contact of K562 and L929 cells or after contact of L929 cells with purified membranes of K562 cells. TNF-alpha or immunologically related proteins, or both, but not Fas-ligand or lymphotoxin, were also accumulated in membranes of K562 and L929 cells shortly after these cells had been allowed to contact each other. The cytotoxic factors expressed by nonlymphoid cells trigger apoptosis of target cells. These observations strongly suggest that nonlymphoid cells possess molecular mechanisms controlling cellular compatibility.

Animals↗
PubMed1997

Spectral studies on the calcium-binding properties of Mts1 protein and its interaction with target protein.

Two calcium-binding sites of the Mts1 protein, a member of S-100 protein family, were distinguished with the Fluo-3 fluorescent technique. The geometric mean of the apparent dissociation constant (Kd) for these two sites is 2.6 microM; the Hill coefficient (nH) is 0.98. In the presence of a novel target protein p37, isolated from the mouse adenocarcinoma cell line CSML-100, Mts1 binds Ca2+ ions with higher affinity and with strong positive cooperativity (Kd = 0.2 microM, nH = 1.91). Interaction of Mts1 with p37 is confirmed by the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Reaction with TNS shows that p37 interacts with the hydrophobic site of Mts1 which is exposed due to the binding of Ca2+ ions.

Animals↗
PubMed1997

Calcium-binding characteristics and conformational changes in metastasin, a member of S-100 protein family.

Investigation of Ca(2+)-binding characteristics of metastasin (Mts-1) by competition with Fluo-3 revealed two types of Ca(2+)-binding sites in Mts-1 with the geometric mean of their dissociation constant (Kd) value of 2.6 microM for the two EF-sites. The Hill coefficient (nH) is 0.98. A substantial increase in the affinity of Mts-1 for Ca2+ and strong cooperative character of binding (kd = 0.2 microM, nH = 1.91) is observed in the presence of the target protein p37 isolated from mouse adenocarcinoma cell lines CSML-100 and CSML-0. Two different hydrophobic sites of binding with the fluorescent probe 2-(p-toludino)naphthalene-6-sulfonate (TNS) per Mts-1 molecule have been determined. The exposure of the hydrophobic binding sites of the first type are shown to be Ca(2+)-dependent and the hydrophobic binding sites of the second type are exposed independently of Ca2+ concentration. A decrease in the number of Ca(2+)-dependent hydrophobic centers in the presence of p37 protein was detected by measurements of TNS fluorescence.

Adenocarcinoma↗
PubMed1996

[Study of calcium-binding properties of the Mts-1 protein and its recombinant analog using the fluorescent probe Fura-2].

A procedure based on the ability of Mts-1 to competitively inhibit the formation of Fura-2 complexes with Ca(2+)-ions is described. It has been shown that Mts-I reversibly inhibits the complex formation between Fura-2 and calcium. The efficiency of natural Mts-I isolated from cells of the CSML-100 line and of its recombinant analog on the formation of the Fura-2+Ca complex is different. The inhibition constant, Ki, for the native protein is equal to 2,4 microM, that for the recombinant form is 8.5 microM. The data obtained are suggestive of posttranslational modification of Mts-I in cells under in vivo conditions.

Calcium↗
PubMed1991

[Oxidative inactivation of angiotensin-converting enzyme].

Hydrogen peroxide inactivates the purified human angiotensin-converting enzyme (ACE) in vitro; the inactivating effect of H2O2 is eliminated by an addition of catalase. The lung and kidney ACE are equally sensitive to the effect of hydrogen peroxide. After addition of oxidants (H2O2 alone or H2O2 + ascorbate or H2O2 + Fe2+ mixtures) to the membranes or homogenates of the lung, the inactivation of membrane-bound ACE is far less pronounced despite the large-scale accumulation of lipid peroxidation products. The marked inactivation of ACE in the membrane fraction (up to 55% of original activity) was observed during ACE incubation with a glucose:glucose oxidase:Fe2+ mixture. Presumably the oxidative potential of H2O2 in tissues in consumed, predominantly, for the oxidation of other components of the membrane (e.g., lipids) rather than for ACE inactivation.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1988

Atriopeptin 2 is hydrolysed by cardiac but not pulmonary isozyme of angiotensin-converting enzyme.

Hydrolysis of Bz-Gly-Ser-Phe-Arg, C-terminal fragment of atriopeptin 2, by human cardiac angiotensin-converting enzyme has been studied. The KM for the reaction was 10(-4) M. The effect of concentration of NaCl on activity of cardiac angiotensin-converting enzyme has been determined, which allowed to regard Bz-Gly-Ser-Phe-Arg as bradykinin-like substrates. It was demonstrated that cardiac, but not pulmonary isozyme of angiotensin-converting enzyme specifically hydrolyses atriopeptin 2.

Atrial Natriuretic Factor↗
PubMed1987

Affinity chromatography and some properties of the angiotensin-converting enzyme from human heart.

Human heart angiotensin-converting enzyme has been purified by affinity chromatography on immobilized N-[1(S)-carboxy-5-aminopentyl]-Gly-Gly. The isolation procedure permitted a 1650-fold-purified enzyme to be obtained. The specific activity of angiotensin-converting enzyme was 38 units per mg protein. The molecular weight of enzyme determined by polyacrylamide gel electrophoresis in denaturing conditions was 150,000. The isoelectric point (5.3) of the enzyme was determined by chromatofocusing. The Km values of the enzyme for Bz-Gly-His-Leu and angiotensin I are 1.2 mM and 10 microM, respectively. Substrate inhibition of heart angiotensin-converting enzyme with a K's of 14 mM has been shown. The human heart enzyme is inhibited by SQ 20881 (IC50 = 40 nM). It was shown that NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- are activators of the heart angiotensin-converting enzyme, whereas CH3COONa and NaNO3 have no effect on a catalytic activity of the enzyme.

Angiotensin I↗
PubMed1987

Monoclonal antibody to human lung angiotensin-converting enzyme.

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunized with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by enzyme-linked immunosorbent assay and that of soluble ACE by an immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not affect ACE activity. We believe this antibody will be useful not only for immunoassay and immunopurification of ACE, but also as a tool for the investigation of the tissue distribution of the enzyme as well as for the study of the structure and mechanism of action of ACE.

Antibodies, Monoclonal↗
PubMed1987

[Various properties of angiotensin-converting enzyme from the seal Phoca groenlandica].

It was found that the molecular mass of the angiotensin-converting enzyme from seal (Phoca groenlandica) lungs determined by electrophoresis in 7.5% PAAG in the presence of sodium dodecyl sulfate is 150 kD. The enzyme has a pH optimum with respect to hippuryl-L-histidyl-L-leucine at 7.3--7.5; KM is 1.2 mM. The enzyme is inhibited by the substrate to form a nonproductive ES2 complex with the dissociation constant (Ks') of 4.8 mM. The activation of the seal angiotensin-converting enzyme in the presence of NaCl was studied. The bradykinin-potentiating factor (SQ 20881) inhibits the seal enzyme with a high efficiency (IC50 = 2.5.10(-8) M). Monoclonal antibodies to the angiotensin-converting enzyme from human lungs do not interact with its seal lung counterpart, which points to the species specificity of the angiotensin-converting enzyme.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1987

[Monoclonal antibodies to the angiotensin-converting enzyme from the human lung].

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunised with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by ELISA; that of soluble ACE--by immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not effect ACE activity. This antibody is believed useful not only for immunoassay and immunopurification of ACE, but also as a tool for investigation of enzyme distribution in tissue as well as for studying the structure and mechanism of ACE action.

Animals↗
PubMed1986

[Isolation and properties of the angiotensin-converting enzyme from human lungs].

Using chromatofocusing, an angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human lung. The procedure allows for 24 300-fold purification of the enzyme. The enzyme specific activity is 36.3 u. per mg protein; Mr as determined by polyacrylamide gel electrophoresis is 150 000. The lung enzyme after solubilization by trypsin treatment was found to be heterogeneous. Four isoforms of the enzyme with pI 5.3, 4.9, 4.8 and 4.6 were identified. The pH-optimum for the enzyme with respect to hippuryl-L-histidyl-L-leucine hydrolysis lies at 8.3; Km = 2.8 mM. The effect of Cl- on the enzyme activity was studied. It was found that the bradykinin-potentiating factor (SQ 20 881) inhibits the human lung angiotensin-converting enzyme (I50 = 1.6 X 10(-8) M).

Angiotensin-Converting Enzyme Inhibitors↗