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1.
Moore AT Rankin KE von Dassow G Peris L Wagenbach M Ovechkina Y Andrieux A Job D Wordeman L 《The Journal of cell biology》2005,169(3):391-397
MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs. 相似文献
2.
Malygin EG Ovechkina LG Zinov'ev VV Lindstrem UM Reich NO 《Molekuliarnaia biologiia》2001,35(1):42-51
Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer oligonucleotide duplex containing the palindrome recognition site GGATCC was studied by methods of steady-state and presteady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaric aldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward a 20-mer oligonucleotide substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower then the concentrations of substrates. The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s-1. The Km values for the duplex and SAM are 0.35 and 1.6 microM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer oligonucleotide duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s-1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in the course of the enzyme single turnover, which coincides with the earlier data on EcoRI MTase. Regardless of the order of the enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme-substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding of DNA. In gel retardation experiments, there was no stoichiometrically simple complexes with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state. 相似文献
3.
Malygin E. G. Ovechkina L. G. Zinoviev V. V. Lindstrem U. M. Reich N. O. 《Molecular Biology》2001,35(1):35-44
Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer duplex containing a palindromic recognition site GGATCC was studied by methods of steady-state and pre-steady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaraldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward the 20-mer substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower than the concentrations of substrates. The k
cat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s–1. The K
M values for the duplex and SAM are 0.35 and 1.6 M, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s–1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in a single turnover of the enzyme, which coincides with the earlier data on EcoRI MTase. Regardless of the order of enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme–substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding with DNA. In gel retardation experiments, there was no stoichiometrically simple complex with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state. 相似文献
4.
Andrews PD Ovechkina Y Morrice N Wagenbach M Duncan K Wordeman L Swedlow JR 《Developmental cell》2004,6(2):253-268
Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore. 相似文献
5.
The structural and catalytic properties of the phage T4 DNA-(adenine-N6)-methyltransferase (EC 2.1.1.72) were studied at different enzyme-substrate concentration ratios by chemical cross-linking of the protein subunits and by measuring the presteady state kinetics of the reactions. Various structural states of the methyltransferase were correlated with its catalytic activity, and it was shown that the oligomeric forms of the enzyme are catalytically active but are characterized by the reaction parameters different from those of the monomer. 相似文献
6.
Yu. G. Maksimova V. A. Demakov A. Yu. Maksimov G. V. Ovechkina G. A. Kovalenko 《Applied Biochemistry and Microbiology》2010,46(4):379-384
The nitrile hydratase isolated from Rhodococcus ruber strain gt1, displaying a high nitrile hydratase activity, was immobilized on unmodified aluminum oxides and carbon-containing
adsorbents, including the carbon support Sibunit. The activity and operational stability of the immobilized nitrile hydratase
were studied in the reaction of acrylonitrile transformation into acrylamide. It was demonstrated that an increase in the
carbon content in the support led to an increase in the amount of adsorbed enzyme and, concurrently, to a decrease in its
activity. The nitrile hydratase immobilized on Sibunit and carbon-containing aluminum α-oxide having a “crust” structure displayed
the highest operational stability in acrylonitrile hydration. It was shown that the thermostability of adsorbed nitrile hydratase
increased by one order of magnitude. 相似文献
7.
The effect of alternating magnetic field (8 Hz, 5 microT) on lipid peroxidation, thiol-disulfide exchange, the antioxidant system, and energy metabolism in liver of animals was studied. It was found that metabolic changes caused by the application of magnetic field vary in animals with different behavior in the "open field". Statistically significant changes in the activity of some enzymes involved in the antioxidant system and thiol-disulfide exchange were revealed. 相似文献
8.
Yu. A. Nikolaev V. K. Plakunov N. A. Voronina N. V. Nemtseva A. O. Plotnikov O. A. Gogoleva M. E. Murav’eva G. V. Ovechkina 《Microbiology》2008,77(1):78-83
The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect. 相似文献
9.
Yu. G. Maksimova A. Yu. Maksimov V. A. Demakov K. V. Kozlov G. V. Ovechkina V. F. Olontsev 《Applied Biochemistry and Microbiology》2011,47(7):681-687
Cells of the Pseudomonas fluorescens strain C2 containing nitrilase and Rhodococcus ruber strain gt1 with nitrile hydratase activity have been immobilized by the use of adsorption on fibrous carbon materials. It
has been shown that the maximum adsorption value of Rhodococcus cells is higher than that in pseudomonades, reaching 21 mg
of dry cells/1 g of the carrier vs. 6 mg, respectively. Cell adsorption, compared to cell suspension, gives a significant
rise in nitrilase activity (by 7.4 times, using Ural TM-4 as the carrier) and in the stability of nitrile hydratase activity
(5 reaction cycles without loss of activity, using Carbopon-B-active). Immobilized biocatalysts were also obtained by cell
growth from Ps. fluorescens strain C2 and Rhodococcus ruber strain gt1 on fibrous carbon adsorbents. Biocatalyst productivity was higher for both strains when the carbonized material
Ural TM-4 was used as the carrier. 相似文献
10.
Yu. G. Maksimova D. M. Vasil’ev A. S. Zorina G. V. Ovechkina A. Yu. Maksimov 《Applied Biochemistry and Microbiology》2018,54(2):173-178
The ability of the Alcaligenes faecalis 2 strain to utilize acrylamide and acrylic acid upon cultivation with these compounds as the only sources of carbon and energy has been investigated. Complete utilization of the acrylic acid present in the medium at concentrations below 0.113 g/L was observed by cultivation day 5, at a concentration of 0.225 g/L by day 7, and at a concentration of 0.45 g/L by day 17. Complete utilization of the acrylamide present in the medium at concentrations below 0.4 g/L was observed by day 5, at a concentration of 0.9 g/L by day 7, and at a concentration of 1.8 g/L by day 20. Importantly, bacterial growth did not start before complete transformation of acrylamide into acrylic acid. The rate of acrylamide transformation by growing bacteria and a cell suspension in the stationary growth phase amounted to 12.5 mg/L h at a cell concentration of 610 mg/L and 300 mg/L h, at a concentration of 1500 mg/L. A. faecalis 2 cells immobilized on BVV-22 basalt fibers and Carbopon-B-aktiv at concentrations of 3000 and 800 mg dry cells/L, respectively, transformed acrylamide at a rate of 1200 mg/L h. 相似文献