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Konstantin N. Belosludtsev Natalia V. Belosludtseva Eugeny Yu Talanov Kirill S. Tenkov Vlada S. Starinets Alexey V. Agafonov Lyubov L. Pavlik Mikhail V. Dubinin 《生物化学与生物物理学报:生物膜》2019,1861(1):288-297
The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V3 and VDNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V2. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H2O2 production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca2+-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca2+/Pi-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca2+ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K+ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K+ buffer and DNP-induced K+ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed. 相似文献
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Biotransformations using prokaryotic P450 monooxygenases 总被引:5,自引:0,他引:5
Recent studies on microbial cytochrome P450 enzymes have covered several new areas. Advances have been made in structure-function analysis and new non-enzymatic/electrochemical systems for the replacement of NAD(P)H in biocatalysis have been developed. Furthermore, the properties of some enzymes have been re-engineered by site-directed mutagenesis or by methods of directed evolution and new P450s have been functionally expressed and characterized. It is thought that a combination of these approaches will facilitate the use of isolated P450 monooxygenases in biocatalysis. 相似文献
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The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation. 相似文献
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In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human
cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution
procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized
cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing
human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment;
(4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate
in a resin; and (5) preservation of antigens for sensitive immunogold labeling. 相似文献
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The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is
responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1
and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates.
In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with
the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically
as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to
hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained
at 51°C, and the half-life at 50°C was with 100 min more than ten times longer than that of CYP102A1 (8 min). 相似文献
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Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants 下载免费PDF全文
Feenstra KA Starikov EB Urlacher VB Commandeur JN Vermeulen NP 《Protein science : a publication of the Protein Society》2007,16(3):420-431
Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants--F87A, L188Q/A74G, and F87V/L188Q/A74G--were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (omega-1, omega-2, omega-3) but not the terminal (omega) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen-atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios. 相似文献
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Milka Elshishka Stela Lazarova Georgi Radoslavov Petar Hristov Vlada K. Peneva 《ZooKeys》2015,(511):25-68
The taxonomic position of two antarctic dorylaimid species Amblydorylaimus
isokaryon (Loof, 1975) Andrássy, 1998 and Pararhyssocolpus
paradoxus (Loof, 1975), gen. n., comb. n. are discussed on the basis of morphological, including SEM study, morphometric, postembryonic and sequence data of 18S rDNA and the D2-D3 expansion fragments of large subunit rDNA. The evolutionary trees inferred from 18S sequences show insufficient resolution to determine the assignment of the two species to particular families, moreover Pararhyssocolpus
paradoxus
gen. n., comb. n. (=Rhyssocolpus
paradoxus) previously regarded as a member of Nordiidae or Qudsianematidae, showed distant relationship both to Rhyssocolpus
vinciguerrae and Eudorylaimus spp. The phylogram inferred from 28S sequences revealed that Amblydorylaimus
isokaryon is a member of a well-supported group comprised of several Aporcelaimellus spp., while, no close relationships could be revealed for the Pararhyssocolpus
paradoxus
gen. n., comb. n. to any nematode genus. On the basis of molecular data and morphological characteristics, some taxonomic changes are proposed. Amblydorylaimus
isokaryon is transferred from family Qudsianematidae to family Aporcelaimidae, and a new monotypic genus Pararhyssocolpus
gen. n. is proposed, attributed to Pararhyssocolpidae
fam. n. The diagnosis of the new family is provided together with emended diagnosis of the genera Amblydorylaimus and Pararhyssocolpus
gen. n. Data concerning distribution of these endemic genera in the Antarctic region are also given. 相似文献
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