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1.
V. N. Stepanenko R. S. Esipov A. I. Miroshnikov V. L. Andronova G. A. Galegov M. V. Yasko A. A. Gus’kova A. Yu. Skoblov Yu. S. Skoblov 《Russian Journal of Bioorganic Chemistry》2011,37(4):436-440
Thymidine kinase UL23 gene (EC 2.7.1.145) from the L2 acyclovir-sensitive strain of herpes simplex virus type 1 was cloned and expressed in E. coli. The enzyme was purified by chromatography to the purity of 90% according to PAG electrophoresis data. The Michaelis constants
for the reactions with thymidine and acyclovir were determined. The enzyme was found to phosphorylate modified nucleosides,
particularly 3′-deoxythymidine, 3′-deoxy-2′,3′-didehydrothymidine, 2′,3′-dideoxycytidine, 9-[(hydroxyethyl)methyl]guanine,
E-5-(2-bromovinyl-2′-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, 2′,3′-dideoxydehydrothymidine, β-L-2′,3′-dideoxy-3′-thiacytidine, and 3′-fluoro-3′-deoxythymidine. Some properties of the purified enzyme were compared with
those of thymidine kinases of other herpes simplex virus strains. It was shown that acyclovir H-phosphonate inhibited the
enzyme. 相似文献
2.
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
3.
Paul V. Bernhardt Piao Chin Philip C. Sharpe Jing-Yan C. Wang Des R. Richardson 《Journal of biological inorganic chemistry》2005,10(7):761-777
The search for orally effective drugs for the treatment of iron overload disorders is an important goal in improving the health
of patients suffering diseases such as β-thalassemia major. Herein, we report the syntheses and characterization of some new
members of a series of N-aroyl-N′-picolinoyl hydrazine chelators (the H2IPH analogs). Both 1:1 and 1:2 FeIII:L complexes were isolated and the crystal structures of Fe(HPPH)Cl2, Fe(4BBPH)Cl2, Fe(HAPH)(APH) and Fe(H3BBPH)(3BBPH) were determined (H2PPH=N,N′-bis-picolinoyl hydrazine; H2APH=N-4-aminobenzoyl-N′-picolinoyl hydrazine, H23BBPH=N-3-bromobenzoyl-N′-picolinoylhydrazine and H24BBPH=N-(4-bromobenzoyl)-N′-(picolinoyl)hydrazine). In each case, a tridentate N,N,O coordination mode of each chelator with Fe was observed. The FeIII complexes of these ligands have been synthesized and their structural, spectroscopic and electrochemical characterization
are reported. Five of these new chelators, namely H2BPH (N-(benzoyl)-N′-(picolinoyl)hydrazine), H2TPH (N-(2-thienyl)-N′-(picolinoyl)-hydrazine), H2PPH, H23BBPH and H24BBPH, showed high efficacy at mobilizing 59Fe from cells and inhibiting 59Fe uptake from the serum Fe transport protein, transferrin (Tf). Indeed, their activity was much greater than that found for
the chelator in current clinical use, desferrioxamine (DFO), and similar to that observed for the orally active chelator,
pyridoxal isonicotinoyl hydrazone (H2PIH). The ability of the chelators to inhibit 59Fe uptake could not be accounted for by direct chelation of 59Fe from 59Fe–Tf. The most effective chelators also showed low antiproliferative activity which was similar to or less than that observed
with DFO, which is important in terms of their potential use as agents to treat Fe-overload disease. 相似文献
4.
5.
John L. Lewin David E. Heppner Christopher J. Cramer 《Journal of biological inorganic chemistry》2007,12(8):1221-1234
The bis(μ-oxo)/μ-η2:η2-peroxo equilibria for seven supported Cu2O2 cores were studied with different hybrid and nonhybrid density functional theory models, namely, BLYP, mPWPW, TPSS, TPSSh,
B3LYP, mPW1PW, and MPW1K. Supporting ligands 3,3′-iminobis(N,N-dimethylpropylamine), N,N,N′,N′,N″-pentamethyldipropylenetriamine, N-[2-(pyridin-2-yl)ethyl]-N,N,N′-trimethylpropane-1,3-diamine, bis[2-(2-pyridin-2-yl)ethyl]methylamine, bis[2-(4-methoxy-2-pyridin-2-yl)ethyl]methylamine,
bis[2-(4-N,N-dimethylamino-2-pyridin-2-yl)ethyl]methylamine, and 1,4,7-triisopropyl-1,4,7-triazacyclononane were chosen on the basis of
the availability of experimental data for comparison. Density functionals were examined with respect to their ability accurately
to reproduce experimental properties, including, in particular, geometries and relative energies for the bis(μ-oxo) and side-on
peroxo forms. While geometries from both hybrid and nonhybrid functionals were in good agreement with experiment, the incorporation
of Hartree–Fock (HF) exchange in hybrid density functionals was found to have a large, degrading effect on predicted relative
isomer energies. Specifically, hybrid functionals predicted the μ-η2:η2-peroxo isomer to be too stable by roughly 5–10 kcal mol−1 for each 10% of HF exchange incorporated into the model. Continuum solvation calculations predict electrostatic effects to
favor bis(μ-oxo) isomers by 1–4 kcal mol−1 depending on ligand size, with larger ligands having smaller differential solvation effects. Analysis of computed molecular
partition functions suggests that nonzero measured entropies of isomerization are likely to be primarily associated with interactions
between molecular solutes and their first solvation shell.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
7.
Cyanobacteria produce some carotenoids. We identified the molecular structures, including the stereochemistry, of all the
carotenoids in the terrestrial cyanobacterium, Nostoc commune NIES-24 (IAM M-13). The major carotenoid was β-carotene. Its hydroxyl derivatives were (3R)-β-cryptoxanthin, (3R,3′R)-zeaxanthin, (2R,3R,3′R)-caloxanthin, and (2R,3R,2′R,3′R)-nostoxanthin, and its keto derivatives were echinenone and canthaxanthin. The unique myxol glycosides were (3R,2′S)-myxol 2′-fucoside and (2R,3R,2′S)-2-hydroxymyxol 2′-fucoside. This is only the second species found to contain 2-hydroxymyxol. We propose possible carotenogenesis
pathways based on our identification of the carotenoids: the hydroxyl pathway produced nostoxanthin via zeaxanthin from β-carotene,
the keto pathway produced canthaxanthin from β-carotene, and the myxol pathway produced 2-hydroxymyxol 2′-fucoside via myxol
2′-fucoside. This cyanobacterium was found to contain many kinds of carotenoids and also displayed many carotenogenesis pathways,
while other cyanobacteria lack some carotenoids and a part of carotenogenesis pathways compared with this cyanobacterium. 相似文献
8.
A. M. Varizhuk S. V. Kochetkova N. A. Kolganova E. N. Timofeev V. L. Florentiev 《Russian Journal of Bioorganic Chemistry》2010,36(4):528-531
Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where
X = H, (S)-CH3, and (R)-CH3. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation
of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared
to that of native ones (ΔT
m∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (ΔT
m∼+2°C per modification). 相似文献
9.
10.
Jian Wen Wang Li Ping Zheng Ben Zhang Ting Zou 《Applied microbiology and biotechnology》2009,85(2):285-292
This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide
(NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the
cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N
ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity
of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the
mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and
the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside
elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the
control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway
of the elicitor activity for artemisinin biosynthesis. 相似文献
11.
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
12.
Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins.
In this study, the full-length cDNA and genomic DNA of F3′H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3’′H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis
revealed that IbF3′H was highly homologous with F3′Hs from other plant species. Conserved domain search revealed that IbF3′H was a cytochrome P450 dependent enzyme. Three F3′H-specific
motifs (V75VVAAS80, G427GEK430 and V433DVKG437) were conserved in IbF3′H. Phylogenetic analysis revealed that IbF3′H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3′H gene in the genome of I. batatas. IbF3′H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves.
During storage root formation, IbF3′H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground
organs. IbF3′H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative
analysis implied that the full-length cDNA of IbF3′H could encode a functional protein and had a special catalytic activity of flavonoid 3′-hydroxylase. 相似文献
13.
Adenosine-5′-methylphosphate (MepA) initiates the oligomerization of the 5′-phosphorimidazolide of uridine (ImpU) in the presence
of montmorillonite clay. Longer oligomers form because the 5′-phosphate is blocked with a methyl group that prevents the formation
of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69–84% of the 5–9 charge oligomers, respectively. The
montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA
→ MepA3′pU → MepA3′pU2′pU → MepA3′pU2′pU3′pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation
of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA3′pU and MepA2′pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA3′pA and MepA2′pA the ImpA reacts 5.2 times faster with MepA3′pA. In the competitive reaction of ImpU and ImpA with MepA3′pA and MepA3′pU the elongation proceeds on MepA3′pA 5.6 times more rapidly than with MepA3′pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer
oligomers. The formation of more than two sequential 2′,5′-linkages in the oligomer chain proceeds more slowly than the addition
to a single 2′,5′-link or a 3′,5′-link and either chain termination or elongation by a 3′,5′-linage occurs. The central role
that catalysis may have had in the prebiotic formation of biopolymers is discussed.
Note added in proof: There are errors in the high resolution mass spectral data given in Section 4.2.1. The high resolution mass spectrum found
for the cyclic dimer of UpUp (C-UpUp) was 657.02260. C18H21N4O16P2Na2 requires 657.02232. The high resolution mass spectrum found for the cyclic dimer of ApAp (C-ApAp) was 725.05850. C20H22N10O12P2Na3 requires 725.05839. 相似文献
14.
Seidel V Windhövel J Eaton G Alfermann AW Arroo RR Medarde M Petersen M Woolley JG 《Planta》2002,215(6):1031-1039
Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological
studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA
ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding
experiments were performed with [2-13C]3′,4′-dimethoxycinnamic acid, [2-13C]3′,4′-methylenedioxycinnamic acid (MDCA), [2-13C]3′,4′,5′-trimethoxycinnamic acid, [2-13C]sinapic acid, [2-13C]- and [2,3-13C2]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from
ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX.
These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears
that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX.
Electronic supplementary material to this article is available at and is accessible for authorized users.
Electronic Publication 相似文献
15.
Huston James S. Adams Gregory P. McCartney John E. Tai Mei-Sheng Hudziak Robert M. Oppermann Hermann Stafford Walter F. Liu Sen Fand Irwin Apell Gerald Laminet Axel Bookman Michael A. Houston L. L. Weiner Louis M. 《Cell biochemistry and biophysics》1994,24(1-3):267-278
This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused
to the sFv carboxyl terminus to facilitate disulfide bonding or specific crosslinking of this sFv′ to make divalent (sFv′)2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv′ and (sFv′)2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv′)2 demonstrated by its complex formation with ECD. The tumor binding properties of125I-labeled anti-c-erbB-2 741F8 sFv, sFv′, and (sFv′)2 were compared with radiolabeled antidigoxin 26-10 sFv′ and (sFv′)2 controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing
SK-OV-3 tumors (which overexpress c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ
ratios demonstrated the 741F8 sFv, sFv′, and (sFv′)2 had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial
pooling in tumors observed with the 26-10 sFv, sFv′, and (sFv′)2, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked
741F8 (sFv′)2 having Gly4Cys tails than for monovalent 741F8 sFv′ or Fab. The superior properties of the 741F8 (sFv′)2 in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting. 相似文献
16.
The number of phosphate groups in the 5′,5′-polyphosphate bridge of mRNA-cap dinucleotide analogues affects kinetics of long-range
electron transfer (ET) responsible for 3-methylbenzimidazole (m3B) fluorescence quenching in model dinucleotides. For instance, 3-methylbenzimidazolyl(5′-5′)guanosine dinucleotides (m3Bp
n
G, n = 2, 3, 4) having m3B donor, 5′-5′ polyphosphate bridge, and guanine (G) acceptor, exhibit exponential dependence of the ET rate on the number
of phosphates, i.e. donor–acceptor distance. Involvement of the 5′-5′ polyphosphate bridge in the ET is strongly indicated
by lack of m3B-G stacking effect on the exponential factor, which is the same at 20°C, where m3B-G intramolecular stacking dominates, as that at 75°C where stacking–unstacking equilibrium is shifted in favour of the unstacked
structure. 相似文献
17.
The removal of endocrine-disrupting compounds (EDCs) by lignin peroxidase from white-rot fungus Phanerochaete sordida YK-624 (YK-LiP1) was investigated. Five endocrine disruptors, p–t-octylphenol (OP), bisphenol A (BPA), estrone (E1), 17β-estradiol (E2), and ethinylestradiol (EE2) were eliminated by YK-LiP1 more effectively than lignin peroxidase from P. chrysosporium (Pc-LiP), and OP and BPA were disappeared almost completely in the reaction mixture containing YK-LiP1 after a 24-h treatment.
Particularly, the removal of estrogenic activities of E2 and EE2, which show much higher estrogenic activities than other EDCs such as BPA and OP, were removed following 24-h treatment with
YK-LiP1. Moreover, 5,5′-bis(1,1,3,3-tetramethylbutyl)-[1,1′-biphenyl]-2,2′-diol and 5,5′-bis-[1-(4-hydroxy-phenyl)-1-methyl-ethyl]-biphenyl-2,2′-diol
were identified as the main metabolite from OP or BPA, respectively. These results suggest that YK-LiP1 is highly effective
in removing of EDCs by the oxidative polymerization of these compounds. 相似文献
18.
Jinwook Seo Suk Weon Kim Jonghyun Kim Hyun Wook Cha Jang R. Liu 《Journal of Plant Biology》2007,50(6):626-631
Theflavonoid 3′,5′-hydroxylase (F3′,5′H) gene, derived from petunia, was introduced into chrysanthemum tissues by Agrobacterium-mediated genetic transformation.
Cotyledon expiants were co-cultured withA. tumefaciens LBA 4404 harboring the vector pMBP that carriesF3′,5′H under the control of the CaMV 35S promoter andnptll as a selectable marker gene. After 72 h of co-cultivation, the expiants were placed on an MS medium supplemented with 4 mg
L-1 BA, 0.1 mg L-1 NAA, 400 mg L-1 carbenicillin, and 100 mg L-1; kanamycin. After 4 weeks, kanamycin-resistant adventitious shoots had developed at a frequency of 6.3%. These shoots were
then rooted and acclimatized in potting soil. Integration ofF3′,5′H into the plant genome was confirmed by Southern blot analysis. Flower buds that had red petals did not differ between the
transgenic and the wild-type plants. However, petal color did change from red to bright orange to yellow when the buds developed
into fully opened flowers on the transgenics. Spectrometric analysis revealed that the content of flavonoid compounds was
more rapidly reduced in the transgenic petals as floral development proceeded. RT-PCR analysis showed thatF3′,5′H andflavonoid 3′hydroxylase (F3′H) were expressed simultaneously in the transgenic plants. Therefore, we suggest that this more rapid change in petal color
results from 1) competition between levels of transgenicF3′,5′H and endogenousF3′H, each of which uses the same substrate in the flavonoid biosynthetic pathway and 2) the intrinsic substrate specificity of
chrysanthemumDFR (dihydroflavonol 4-reductase). 相似文献
19.
The optical activity induced on binding the drug netrospin (NET) in the minor groove of DNA is studied in five oligonucleotides
(OGNs) as a function of (1) the size of the binding site in (5′-(GC)2AATT(GC)2-3′)2 (OGN 1a) versus (5′-(GC)2AAATTT(GC)2-3′)2 (OGN 1b) and (2) the distance between two AATT binding sites in (5′-(GC)2AATT(GC)
x
AATT(GC)2-3′)2, with x = 1, 2, or 3 (OGNs 2a, b, c, respectively). NET binding is monitored via the induced circular dichroism (CD) at ~315 nm,
where the nucleic acids are optically inactive. The CD titrations, fit to a tight binding model, yield lower limits for the
binding constant, Ka, ≥8 × 107 M−1 for OGN 1a and ≥2 × 108 M−1 for OGNs 2a, b, c in 1 mM buffer. In 100 mM buffer, tight binding occurs in all five OGNs with Ka ≥ 8 × 107 M−1 for OGN 1a and ≥1 × 108 M−1 for OGNs 1b and 2a, b, c. In contrast, the elongated AAATTT binding site of OGN 1b results in weak binding of NET in 1 mM
buffer, where competing electrostatic interactions with the solvent environment are lower. In the constructs with two binding
sites, the increase in flexibility introduced by intervening GC base pairs does not induce co-operative binding, although
differences in the number of binding sites, n (2.05–2.65), indicate that there may be differences in the way NET is bound in OGNs 2a, b, c. In addition, the large shifts
in the absorption spectra induced in bound versus free NET, and effects on the CD spectral bands at higher energy, are discussed
in terms of electrostatic and excitonic interactions. 相似文献
20.
Müller F Aschenbach JR Gäbel G 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2000,170(4):337-343
This study sought to investigate effects of short-chain fatty acids and CO2 on intracellular pH (pHi) and mechanisms that mediate pHi recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pHi was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein
acetoxymethyl ester (BCECF/AM). The resting pHi in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution,
a NH4
+/NH3-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pHi recovery from an NH4
+/NH3-induced acid load by 58% and 70%, respectively. pHi recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM),
respectively. Changing from HEPES- (20 mM) to CO2/HCO3
−-buffered (5%/20 mM) solution caused a rapid decrease of pHi (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked
the pHi recovery by 88%. The results indicate that intracellular acidification by butyrate and CO2 is effectively counter-regulated by an Na+/H+ exchanger and by DIDS-sensitive, HCO3
−-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance
for maintaining the pHi homeostasis of ruminal epithelial cells.
Accepted: 8 March 2000 相似文献