共查询到20条相似文献,搜索用时 15 毫秒
1.
M. V. Tarasova V. V. Kuznetsov N. A. Netesova D. A. Gonchar S. Kh. Degtyarev 《Moscow University Biological Sciences Bulletin》2011,66(2):76-78
DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases.
Gene M1.BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers.
It has been shown that M1.BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA
methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min−1. K
mphage is λ DNA—0.053 ± 0.007 μM, and K
mSAM is 5.1 ± 0.3 μM. 相似文献
2.
3.
The statistical correlation of nucleotides in a DNA sequence is described by a set of redundanciesD
1,D
2,D
3,... By calculation of {D
n} of 2341 coding regions of nucleic acid sequences it is demonstrated that about 2/3 of sequences has correlation length ≤2,
10% of sequences—correlation with 3-periodicity and others—long range aperiodic correlations. The implications of the results
from the interactions of random mutation and natural selection are discussed briefly.
Project supported by National Science Foundation of China. 相似文献
4.
Rücker B Almeida ME Libermann TA Zerbini LF Wink MR Sarkis JJ 《Molecular and cellular biochemistry》2007,306(1-2):247-254
In the present study we investigate the biochemical properties of the members of NPP family in synaptosomes prepared from
rat heart left ventricles. Using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation dependence
and the K
M
value corresponded to 91.42 ± 13.97 μM and the maximal velocity (V
max
) value calculated was 63.79 ± 3.59 nmol p-nitrophenol released/min/mg of protein (mean ± SD, n = 4). Levamisole (1 mM), was ineffective as inhibitor of p-Nph-5′-TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis
of p-Nph-5′-TMP by about 46%. Sodium azide (10 and 20 mM) and gadolinium chloride (0.3 and 0.5 mM), E-NTPases inhibitors, had
no effects on p-Nph-5′-TMP hydrolysis. RT-PCR analysis of left ventricle demonstrated the expression of NPP2 and NPP3 enzymes, but excluded
the presence of NPP1 member. By quantitative real-time PCR we identified the NPP3 as the enzyme with the highest expression
in rat left ventricle. The demonstration of the presence of the E-NPP family in cardiac system, suggest that these enzymes
could contribute with the fine-tuning control of the nucleotide levels at the nerve terminal endings of left ventricles that
are involved in several cardiac pathologies. 相似文献
5.
A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin
3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate
specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable
k
cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K
m value for 1 (3.89 × 10−5 M), which is lower than that for 2 (1.38 × 10−4 M), renders the k
cat/K
m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K
m value for UDP-glucose (6.18 × 10−3 M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10−3 M) with saturated 1, the k
cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center.
K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work. 相似文献
6.
Tarasova MV Kuznetsov VV Netesova NA Gonchar DA Degtyarev SKh 《Biochemistry. Biokhimii?a》2010,75(12):1484-1490
A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5′-CCGC-3′) comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature
optimum of 30°C and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence
5′-CCGC-3′. The kinetic parameters of M1.BspACI DNA methylation are as follows: K
m for phage λ DNA is 0.053 μM and K
m for S-adenosyl-L-methionine is 5.1 μM. The catalytic constant (k
cat) is 0.095 min−1. 相似文献
7.
Masłyk M Kochanowicz E Zieliński R Kubiński K Hellman U Szyszka R 《Molecular and cellular biochemistry》2008,312(1-2):61-69
Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid
sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification
and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant
CK2α (K
m 0.35 μM) and CK2α′ (K
m 0.18 μM) as well as CK2 holoenzyme (K
m 1.1 μM). Different K
m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor
Svf1. Reconstitution of α′2ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit.
This effect was not observed in the case of α2ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments).
Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and
TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K199EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED248 of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation
of cell survival pathways. 相似文献
8.
M. A. Ivanov G. S. Ludva A. V. Mukovnya S. N. Kochetkov V. L. Tunitskaya L. A. Alexandrova 《Russian Journal of Bioorganic Chemistry》2010,36(4):488-496
4′-Fluoro-2′,3′-O-isopropylidenecytidine was synthesized by the treatment of 5′-O-acetyl-4′-fluoro-2′,3′-O-isopropylideneuridine with triazole and 4-chlorophenyl dichlorophosphate followed by ammonolysis. The interaction of 4′-fluoro-2′,3′-O-isopropylidenecytidine with hydroxylamine resulted in 4′-fluoro-2′,3′-O-isopropylidene-5′-O-acetyl-N
4-hydroxycytidine. The removal of the 2′,3′-O-isopropylidene groups led to acetyl derivatives of 4′-fluorouridine, 4′-fluorocytidine, and 4′-fluoro-N
4-hydroxycytidine. 4′-Fluorouridine 5′-O-triphosphate was obtained in three steps starting from 4′-fluoro-2′,3′-O-isopropylideneuridine. 4′-Fluorouridine 5′-O-triphosphate was shown to be an effective inhibitor of HCV RNA-dependent RNA polymerase and a substrate for the NTPase reaction
catalyzed by the HCV NS3 protein, the hydrolysis rate being similar to that of ATP. It could also activate a helicase reaction
with an efficacy of only threefold lower than that for ATP. 相似文献
9.
The stability constants of the 1 : 1 complexes formed between Pb2+ and several simple phosphate monoesters (4-nitrophenyl phosphate, phenyl phosphate, d-ribose 5-monophosphate, n-butyl phosphate) or phosphonate ligands (methylphosphonate, ethylphosphonate) (R-PO2–
3) were determined by potentiometric pH titrations in aqueous solution (25 °C;I=0.1 M, NaNO3). The construction of a log K
P
P
b
b(R-PO3) versus pK
H
H(R-PO3) plot for the mentioned ligand systems results in a straight line on which the data pairs (the corresponding equilibrium constants
were also measured) for uridine 5′-monophosphate (UMP2–) and thymidine 5′-monophosphate (dTMP2–) also fall; this result shows that in the Pb2+ complexes of UMP2– and dTMP2– the nucleobase residues do not interfere, in neither a positive nor a negative way, with the binding of Pb2+ and that the stability of all these complexes is determined by the basicity of the phosph(on)ate group. The mentioned straight-line
correlation (as defined by the least-squares procedure) allowed us to demonstrate (via constants determined now) that the
stability of the Pb2+ complex of cytidine 5′-monophosphate (CMP2–) is also solely determined by the basicity of its phosphate group. A similar evaluation, based on literature data, for the
Pb(HPO4) complex reveals that its stability corresponds closely to the expectations based on the Pb(R-PO3) data, though there is a slight hint that Pb(HPO4) may be somewhat more stable [which would be in agreement with previous observations of other M(HPO4) complexes]; clearly, more such comparisons are possible with the reference line given now. Based on the stability constants
of the monoprotonated Pb(H;CMP)+ complex and the Pb(cytidine)2+ species (which was also measured now), it is concluded that in Pb(H;CMP)+ the proton is located at the phosphate group and Pb2+ mainly at the N3/(C2)O site of the cytosine residue. Regarding nucleic acids in solution, it is further concluded that the
affinity of Pb2+ towards the negatively mono-charged phosphate unit, —O—P(O)2
–—O—, of a nucleic acid backbone is comparable to that of the cytosine moiety, the affinity towards other nucleobase residues
being smaller. This information may prove helpful regarding the properties of lead ribozymes.
Received: 16 April 1999 / Accepted: 2 June 1999 相似文献
10.
V. N. Barai I. A. Severina T. A. Kukharskaya A. I. Zinchenko 《Applied Biochemistry and Microbiology》2000,36(1):4-7
The properties of the phosphatase present in the culture liquid ofSpicaria violacea were investigated. Based on these results, a method for preparative dephosphorylation of calcium salts of 2′(3′)-mononucleotides
was proposed. A 96–98% yield was achieved at a substrate concentration of 100 mg/ml. Mild quantitative hydrolysis of RNA to
nucleosides can be performed by RNA digestion to mononucleotides with Ca2+ followed by the proposed dephosphorylation procedure. 相似文献
11.
12.
Yu. L. Vechtomova T. A. Telegina M. P. Kolesnikov M. S. Kritsky 《Applied Biochemistry and Microbiology》2010,46(3):339-345
Exposure of deaerated folic acid solutions containing an electron donor to UV radiation (310–390 nm, I = 0.4 W m−2) induced formation of dihydrofolic acid (DHFA), a photoexcitation which gave tetrahydrofolic acid (THFA). Only DHFA was formed in the presence of EDTA (E′o = +0.40 V), while the presence of stronger reductants—NADH (E′o = −0.32 V) and boron hydride (E′o = −0.48 V)—induced photoreduction to THFA. It was demonstrated that UV radiation had no effect on the THFA formylation, giving
the coenzyme 5,10-methenyltetrahydrofolic acid and its transformation into another coenzyme, 5-formyltetrahydrofolic acid. 相似文献
13.
Polyclonal catalytic antibodies (abzymes) play an important role in immunology research. In this study, we report polyclonal
antibodies IgYs isolated from chicken egg yolk with hydrolysis activity for the first time. The IgYs were raised in hens using
HNPBV [4-(hydroxy (naphthalen-2-yloxy) phosphoryl) butanoic acid] attached to BSA (Bovine serum albumin) as an immunogen.
Anti-(HNPBV-BSA) IgYs were isolated from yolks of the eggs laid using a two-step salt precipitation and one-step gel filtration
protocol. NA (naphthalen-2-yl acetate) was selected as the substrate and the hydrolysis reaction of the IgYs for it was examined.
The result reveals that the rate of the hydrolysis reaction is higher (K
cat/K
uncat∼2 × 104). The purified IgYs were digested with pepsin and the smaller fragment (Fab′) with specific antigen binding properties was
produced. The research indicates that the enzymatic properties of Fab′ are similar to IgYs. The catalytic activity of the
IgYs was further determined by measuring the rate of hydrolysis of NA in the presence of inhibitor. These findings show that
chicken egg is an excellent donor for polyclonal catalytic antibodies. 相似文献
14.
Role of P2 purinergic receptors in synaptic transmission under normoxic and ischaemic conditions in the CA1 region of rat hippocampal slices 总被引:3,自引:3,他引:0
The role of ATP and its stable analogue ATPγS [adenosine-5′-o-(3-thio)triphosphate] was studied in rat hippocampal neurotransmission
under normoxic conditions and during oxygen and glucose deprivation (OGD). Field excitatory postsynaptic potentials (fEPSPs)
from the dendritic layer or population spikes (PSs) from the soma were extracellularly recorded in the CA1 area of the rat
hippocampus. Exogenous application of ATP or ATPγS reduced fEPSP and PS amplitudes. In both cases the inhibitory effect was
blocked by the selective A1 adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and was potentiated by different ecto-ATPase inhibitors:
ARL 67156 (6-N,N-diethyl-D-β,γ-dibromomethylene), BGO 136 (1-hydroxynaphthalene-3,6-disulfonate) and PV4 [hexapotassium dihydrogen monotitanoundecatungstocobaltate(II)
tridecahydrate, K6H2[TiW11CoO40]·13H2O]. ATPγS-mediated inhibition was reduced by the P2 antagonist suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulfonate]
at the somatic level and by other P2 blockers, PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate) and MRS 2179 (2′-deoxy-N
6-methyladenosine 3′,5′-bisphosphate), at the dendritic level. After removal of both P2 agonists, a persistent increase in
evoked synaptic responses was recorded both at the dendritic and somatic levels. This effect was prevented in the presence
of different P2 antagonists. A 7-min OGD induced tissue anoxic depolarization and was invariably followed by irreversible
loss of fEPSP. PPADS, suramin, MRS2179 or BBG (brilliant blue G) significantly prevented the irreversible failure of neurotransmission
induced by 7-min OGD. Furthermore, in the presence of these P2 antagonists, the development of anoxic depolarization was blocked
or significantly delayed. Our results indicate that P2 receptors modulate CA1 synaptic transmission under normoxic conditions
by eliciting both inhibitory and excitatory effects. In the same brain region, P2 receptor stimulation plays a deleterious
role during a severe OGD insult. 相似文献
15.
Deshmukh SS 《Current microbiology》2007,54(3):186-189
Nuclease Stn α from Streptomyces thermonitrificans hydrolyses DNA and RNA at the rate of approximately 10:l. The optimum pH and temperature for RNA hydrolysis were 7.0 and
45°C. The RNase activity of nuclease Stn α had neither an obligate requirement of metal ions nor was it activated in the presence
of metal ions. The enzyme was inhibited by Zn2+, Mg2+, Co2+, and Ca2+; inorganic phosphate; pyrophosphate; NaCl; KCl; and metal chelators. It was stable at high concentrations of urea but susceptible
to low concentrations of Sodium dodecyl sulfate and guanidine hydrochloride. The rates by which nuclease Stn α hydrolysed
polyribonucleotides occurs in the order of poly A >> RNA >> poly U > poly G > poly C. The enzyme cleaved RNA to 3′ mononucleotides
with preferential liberation of 3′AMP, indicating it to be an adenylic acid preferential endonuclease. 相似文献
16.
Effects of the extracellular Ca2+ concentration ([Ca2+]
o
) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture.
The major resting conductance in the presence of 1 mm Ca2+ was mediated by a Ba2+-sensitive, inwardly rectifying K+ (IRK) current. A rise in [Ca2+]
o
(5–40 mm) inhibited the IRK current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl− (ORCl) current. The activation of the ORCl current developed slowly and needed higher [Ca2+]
o
than that required to inhibit the IRK current. The inhibition of the IRK current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular
application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition,
however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The
activation of the ORCl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca2+ level neither reduced the IRK current nor activated the ORCl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca2+]
o
-induced changes in the IRK and ORCl conductances. These results suggest that high [Ca2+]
o
had a dual action on the membrane conductance of osteoclasts, an inhibition of an IRK conductance and an activation of an ORCl conductance. The two conductances modulated by [Ca2+]
o
may be involved in different phases of bone resorption because they differed in Ca2+ sensitivity, temporal patterns of changes and regulatory mechanisms.
Received: 28 May 1996/Revised: 28 January 1997 相似文献
17.
Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase 总被引:1,自引:0,他引:1
Kazunari Yoshizawa 《Journal of biological inorganic chemistry》1998,3(3):318-324
A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO)
is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol
conversion by the bare FeO+ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH4) complex with an OFe—CH4 bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital
interactions between a C
3v
- or D
2d
-distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered
structure. The generated intermediate involves an HO—Fe—CH3 moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration
that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments
on regioselectivity of enzyme-catalyzed alkane hydroxylations.
Received: 15 September 1997 / Accepted: 20 December 1997 相似文献
18.
Shi H Yin X Wu M Tang C Zhang H Li J 《Journal of industrial microbiology & biotechnology》2012,39(2):347-357
Using 3′ and 5′ rapid amplification of cDNA ends methods, the full-length cDNA sequence encoding an endo-1,4-β-glucanase of Aspergillus usamii E001 (abbreviated as AuCel12A) was amplified from the total RNA. The clone cDNA sequence of the gene encoding the AuCel12A,
named as Aucel12A, is 1,027 bp in length harboring 5′ and 3′ non-coding regions, as well as a 720 bp of open reading frame that encodes a 16-aa
signal peptide, and a 223-aa mature AuCel12A with a theoretical M.W. of 24,294 Da, a calculated pI of 4.15, and one putative N-glycosylation site. The complete DNA sequence of the gene Aucel12A was amplified from the genomic DNA of A. usamii E001 by using the conventional PCR and pUCm-T vector-mediated PCR initially developed in our lab. The clone DNA sequence
is 1,576 bp in length, consisting of a 5′ flanking regulatory region, three exons, and two introns with sizes of 50 and 66 bp.
The cDNA fragment encoding the mature AuCel12A was expressed in a fully active form in Pichia pastoris. One P. pastoris transformant expressing the highest recombinant AuCel12A (rAuCel12A) activity, labeled as P. pastoris GSCel2-1, was chosen for subsequent studies. Integration of the Aucel12A into P. pastoris genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS-PAGE and enzyme activity assays demonstrated that the rAuCel12A, a glycosylated protein with an apparent M.W.
of 27.0 kDa and a carbohydrate content of 4.82%, was secreted into the culture medium. The purified rAuCel12A displayed the
highest activity at pH 5.0 and 60°C. It was highly stable at a pH range of 3.5–7.0, and at a temperature of 55°C or below.
Its activity was not significantly affected by an array of metal ions and EDTA, but inhibited by Ag+, Hg2+ and Fe2+. The K
m and V
max of the rAuCel12A, towards carboxymethylcellulose-Na (CMC-Na) at pH 5.0 and 50°C were 4.85 mg/ml and 160.5 U/mg, respectively. 相似文献
19.
The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed
by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR)
were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate
3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate
and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K
m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and
40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted
as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis. 相似文献
20.
Brij Bhushan Uma Shanker Kamaluddin 《Origins of life and evolution of the biosphere》2011,41(5):469-482
Manganese exists in different oxidation states under different environmental conditions with respect to redox potential. Various
forms of manganese oxides, namely, Manganosite (MnO), Bixbyite (Mn2O3), Hausmannite (Mn3O4) and Pyrolusite (MnO2) were synthesized and their possible role in chemical evolution studied. Adsorption studies of ribose nucleotides (5′-AMP,
5′-GMP, 5′-CMP and 5′-UMP) on these manganese oxides at neutral pH, revealed a higher binding affinity to manganosite (MnO)
compared to the other manganese oxides. That manganese oxides having a lower Mn-O ratio show higher binding affinity for the
ribonucleotides indirectly implies that such oxides may have provided a surface onto which biomonomers could have been concentrated
through selective adsorption. Purine nucleotides were adsorbed to a greater extent compared to the pyrimidine nucleotides.
Adsorption data followed Langmuir adsorption isotherms, and X
m
and K
L
values were calculated. The nature of the interaction and mechanism was elucidated by infrared spectral studies conducted
on the metal-oxide and ribonucleotide-metal-oxide adducts. 相似文献