共查询到20条相似文献,搜索用时 31 毫秒
1.
The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg2+ and H+. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2-disulfonates. Inhibition of malonate transport approaches 100% with IC50 values of 26, 44, and 88 M for DIDS, H2-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl– transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC50 which is severalfold higher than that for malonate. The IC50 for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC50 by about 50%, but under conditions where only the matrix pH is made alkaline the IC50 is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl– channels than previously thought, but also suggest that other Cl– channels may be sensitive to some of the many regulators of IMAC which have been identified. 相似文献
2.
The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg2+, and is stimulated by K+ and other monovalent cations, both organic and inorganic. The activation of ATPase by K+ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K+ concentration. K+, Mg2+-stimulated ATPase activity is strongly inhibited by N,N-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.Abbreviations DCCD
N,N-dicyclohexylcarbodiimide
- DES
diethylstilbestrol
- DTE
dithioerythritol
- Ellmans r
5-5 dithiobis (2 nitrobenzoic) acid
- FC
fusicoccin
- NPA
naphthylphthalamic acid
- OG
octylguanidine
- PCMBS
p-chloromercuribenzensulphonate 相似文献
3.
Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg2+ and in the presence of Mg2+ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl->HCO
3
-
>Br->malate>acetate>SO
4
2-
. For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH
4
+
Na+>K+). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na+ and K+. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in Vmax rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO3 but other inhibitors tested (azide, oligomycin, orthovanadate, K3[Cr(oxalate)6] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATPF
free ATP
- ATPT
total ATP
- BSA
bovine serum albumen
- DCCD
N,N-dicyclohexylcarbodiimide
- DES
diethylstilbestrol
- DNP
2,4-dinitrophenol
- EDAC
ethyl-3-(3-dimethylaminopropyl)carbodiimide
- Km
apparent Michaelis constant
- MgATP
complex of Mg2+ and ATP
- Mg
F
2+
free Mg2+
- Mg
T
2
total Mg2+
- MES
2-(N-Morpholino)ethanesulphonic acid
- Na2EDTA
disodium ethylenediaminetetraacetic acid
- NEM
N-ethylmaleimide
- Pi
inorganic phosphate
- TCA
trichloroacetic acid
- Tris
tris(hydroxymethyl)methylamine
- Vmax
maximum velocity 相似文献
4.
Summary Crude cell-free extracts of Anabaena cylindrica synthesized adenosine-5-phosphosulphate (AP35S) and 3-phosphoadenosine-5-phosphosulphate (PAP35S) from 35SO4
2- in the presence of Mg2+, ATP and inorganic pyrophosphatase. Maximum AP35S and PAP35S were produced at pH 7.15 and 8.05, respectively. APS kinase was detected in the supernatant of crude cell-free extracts by a spectrophotometric procedure. ATP-Sulphurylase had an absolute requirement for Mg2+ and less than 30% AP35S was formed when Mg2+ was replaced by either Mn2+ or Co2+. Nucleotide triphosphates other than ATP and 2-deoxyATP were ineffective in this reaction. Maximum enzyme activity was observed at equimolar concentrations of Mg2+ and ATP and excess of either of these was inhibitory. Other nucleotide triphosphates, like GTP, UTP, CTP, TTP, ITP, or 2-deoxyATP also inhibited the enzyme activity. Inhibition by GTP was competitive with respect to ATP. ATP-sulphurylase activity was not affected by cysteine, methionine or glutathione.Abbreviations APS
adenosine-5-phosphosulphate
- PAPS
3-phosphoadenosine-5-phosphosulphate 相似文献
5.
José A. Teruel José Tudela Francisco Garcia Carmona Juan C. Gomez Fernandez Francisco Garcia Canovas 《Journal of bioenergetics and biomembranes》1987,19(4):383-396
The (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum presents negative cooperativity for the hydrolysis of Mg2+-ATP at different concentration ranges of this substrate. A kinetic model is proposed according to which Mg2+-ATP may bind to three different enzymatic species present during the catalytic cycle, E (K
1=1 µM), EP.Ca2 (K
9=500 µM) and *EP (K
7=20 µM), accelerating the release of Pi. The fact that each of these species has a different affinity for Mg2+-ATP allows a significant enhancement of the rate of Pi release to the medium at the different ranges of Mg2+-ATP concentration where the enzyme shows a kinetic cooperativity. The kinetic analysis of this mechanism yields an equation which is a ratio of two cubic polynomials (3:3 rate equations) with respect to Mg2+-ATP and which may explain the negative cooperativity of the enzyme at different concentration ranges of Mg2+-ATP.Abbreviations: EGTA, ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid; I.U., international units; piruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27); ATP phosphohydrolase (EC 3.8.1.3). 相似文献
6.
The prime plasmalemma ATPase of the halophilic green alga Dunaliella bioculata has been solubilized by Triton X-100 from a plasmalemma-rich membrane fraction and purified by anion-exchange chromatography. Vanadate-sensitive ATPase activity was totally enriched about 230-fold to a specific activity of approx. 250 nkat·mg protein–1. The presence of Mg2+ or Mn2+ is essential for ATP hydrolysis by the enzyme. In addition to an equimolar requirement (11 Mg2+: ATP), there is further stimulation by Mg2+ (up to 20 mM) and by (100 mM) monovalent cations (K+ NH
4
+
>Rb+ -Na+ >Cs+ >Li+-choline+). Most anions have no or little effect. With a molecular mass of about 105 kDa for the single subunit, sensitivity to vanadate and N,N-dicyclohexylcarbodiimide (50% inhibition at about 1 M and 0.3 mM, respectively), strict ATP-specificity, and an acidic pH optimum, this enzyme shows the typical characteristics of the common type of H+-ATPase in the plasmalemma of higher plants and fungi. These results undermine the hypothesis of a wider distribution of a special (high salt) type of plasmalemma ATPase as found in the marine alga Acetabularia.Abbreviations BTP
1,3-bis[tris(hydroxymethyl)-methylamino]propane
- DCCD
N,N-dicyclohexylcarbodiimide
- DES
diethylstilbestrol
- Mega-9
nonanoyl-N-methyl-glucamide
- Mes
N-morpholinoethanesulfonic acid
- Mops
N-morpholinopropanesulfonic acid
- PAGE
polyacrylamide-gel electrophoresis
- PM
plasmalemma-enriched membrane fraction
- SDS
sodium dodecyl sulfate
This work was supported by the Deutsche Forschungsgemeinschaft; we thank Drs. M. Ikeda and D. Oesterhelt (MPI für Biochemie, Martinsried, FRG) for generous and valuable information about their work prior to publication. 相似文献
7.
The association of an ATPase with the yeast peroxisomal membrane was established by both biochemical and cytochemical procedures. Peroxisomes were purified from protoplast homogenates of the methanol-grown yeast Hansenula polymorpha by differential and sucrose gradient centrifugation. Biochemical analysis revealed that ATPase activity was associated with the peroxisomal peak fractions which were identified on the basis of alcohol oxidase and catalase activity. The properties of this ATPase closely resembled those of the mitochondrial ATPase of this yeast. The enzyme was Mg2+-dependent, had a pH optimum of approximately 8.5 and was sensitive to N,N-dicyclohexylcarbodiimide (DCCD), oligomycin and azide, but not to vanadate. A major difference was the apparent K
m for ATP which was 4–6 mM for the peroxisomal ATPase compared to 0.6–0.9 mM for the mitochondrial enzyme.Cytochemical experiments indicated that the peroxisomal ATPase was associated with the membranes surrounding these organelles. After incubations with CeCl3 and ATP specific reaction products were localized on the peroxisomal membrane, both when unfixed isolated peroxisomes or formaldehyde-fixed protoplasts were used. This staining was strictly ATP-dependent; in controls performed i) in the absence of substrate, ii) in the presence of glycerol 2-phosphate instead of ATP, or iii) in the presence of DCCD, staining was invariably absent. Similar staining patterns were observed in subcellular fractions and protoplasts of Candida utilis and Trichosporon cutaneum X4, grown in the presence of ethanol/ethylamine or ethylamine, respectively.Abbreviations MES
2-(N-Morpholino)ethanesulfonic acid
- DCCD
N,N-dicyclohexylcarbodiimide 相似文献
8.
The chloroplast coupling factor (CF1) was dissociated into subunits by the freezing-thawing procedure in the presence of 0.5 M NaBr and the subunit was purified by ion-exchange chromatography on a DEAE-cellulose column. The subunit did not catalyze ATP hydrolysis either in the presence or in the absence of reagents known to activate Mg2+-dependent ATPase activity of CF1. However, it manifested appreciable adenylate kinase-like and ATP-ADP -phosphate exchange activities. The adenylate kinase-like activity only slightly depended on Mg2+ ions. Ethanol, and especially diadenosine pentaphosphate, inhibited the reaction effectively. In contrast, the ATP-ADP exchange activity was Mg2+-dependent. Ethanol and diadenosine pentaphosphate were poor inhibitors. Sulfite, the CF1-ATPase activator, and quercetin, its inhibitor, had a minor effect on catalytic activity of the subunit.Abbreviations CF
chloroplast coupling factor 1
- RBP
carboxylase-ribulose-1,5-bisphosphate carboxylase
- TLC
thin layer chromatography
- MES
morpholinoethane sulfonic acid
- PMSF
phenylmethylsulfonyl fluoride
- AP5A
diadenosine pentaphosphate, P1, P5-bis(5-adenosyl)pentaphosphate
- DCCD
N1N-dicyclohexylcarbodiimide
- SDS
sodium dodecylsulfate
- PAAG
polyacrylamide gel 相似文献
9.
Zn2+ can induce a conformational change of Band 3 with concomitant inhibition of its anion transport activity of human erythrocyte membrane vesicles only from the cytoplasmic side. The Zn2+ inhibition exhibits a dose-dependent manner with an apparent half maximal concentration of 50 M ZnCl2 and can be reversed by 0.5 mM EDTA, but not by 1 mM dithiothreitol. The Zn2+ effect is specific and no similar inhibitory action could be observed by other divalent cations (Cu2+, Mn2+, Mg2+ or Sr2+).Abbreviations DPA
dipicolinic acid
- EITC
eosin 5-isothiocyanate
- DIDS
4,4-diisothiocyanostilbene-2,2-disulfonate
- TES
N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid
- EDTA
ethylendiamine tetraacetic acid
- DTT
dithiothreitol
- NEM
N-ethylmaleimide
- EITC-Band 3
Band 3 labeled with EITC 相似文献
10.
C. van der Drift D. B. Janssen P. M. G. F. van Wezenbeek 《Archives of microbiology》1978,119(1):31-36
ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg2+ ratio of 2. Mn2+ and Ca2+ could replace Mg2+ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N-dicyclohexylcarbodiimide but not by sodium azide, uncouplers or bathophenanthroline.An electrochemical gradient of protons, which was artificially imposed across the membranes of Streptococcus cells by manipulation of either the K+ diffusion potential or the transmembrane pH gradient, led to ATP synthesis. ATP synthesis was abolished by proton conductors, an inhibitor of the ATPase or an increase in the extracellular K+ concentration. A comparison between the phosphate potential and the electrochemical proton gradient showed that the data found are in agreement with a stoichiometry of 2 protons translocated per molecule ATP synthesized.Abbreviations
electrochemical gradient of protons
- DMO
5,5-dimethyl-2,4-oxazolidinedione
- CCCP
carbonylcyanide m-chlorophenylhydrazone
- FCCP
carbonylcyanide p-trifluoromethoxyphenylhydrazone
- DCCD
N,N-dicyclohexylcarbodiimide
- DNP
2,4-dimitrophenol 相似文献
11.
Peter Gegenheimer 《Molecular biology reports》1995,22(2-3):147-150
Chloroplasts of land plants have an active transfer RNA processing system, consisting of an RNase P-like 5 endonuclease, a 3 endonuclease, and a tRNA:CCA nucleotidyltransferase. The specificity of these enzymes resembles more that of their eukaryotic counterparts than that of their cyanobacterial predecessors. Most strikingly, chloroplast RNase P activity almost certainly resides in a protein, rather than in an RNA protein complex as in Bacteria, Archaea, and Eukarya. The chloroplast enzyme may have evolved from a preexisting chloroplast NADP-binding protein. Chloroplast RNase P cleaves pre-tRNA by a reaction mechanism in which at least one of the Mg2+ ions utilized by the bacterial ribozyme RNase P is replaced by an amino acid side chain.Abbreviations pre-tRNA
precursor to tRNA
- pCp
cytidine 5, 3-bisphosphate
- IC50
inhibitor concentration giving 50% inhibition
- GAPDH
glyceraldehyde 3-phosphate dehydrogenase 相似文献
12.
Robert P. Casey Clemens Broger Marcus Thelen Angelo Azzi 《Journal of bioenergetics and biomembranes》1981,13(5-6):219-228
We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H+ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD
N,N-dicyclohexylcarbodiimide
- NCCD
N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide
- Hepes
2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate
- TMPD
N,N,N,N-tetramethylphenylenediamine 相似文献
13.
ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca2+ and Mg2+ in the complete absence of the other cation. Increasing concentrations of Mg2+ produced an apparent inhibition of the Ca2+-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca2+ with a high affinity (K0.5 Ca2+ = 0.41 M) suggesting the presence of a high affinity Ca2+-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca2+ and Mg2+ concentrations and failed to stimulate membrane phosphorylation and Ca2+ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca2+-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP
adenosine 3,5-monophosphate
- CaM
calmodulin
- CDTA
trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate
- EDTA
ethylene-diaminetetraacetate
- EGTA
ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate
- PLB
phospholamban
- SR
sarcoplasmic reticulum
- TFP
trifluoperazine 相似文献
14.
Fluoride and beryllium interact with the (Na + K)-dependent ATPase as analogs of phosphate 总被引:5,自引:0,他引:5
Joseph D. Robinson Richard L. Davis Marcia Steinberg 《Journal of bioenergetics and biomembranes》1986,18(6):521-531
Fluoride irreversibly inhibits the (Na + K)-ATPase, and this inactivation requires divalent cations (Mg2+, Mn2+, or Ca2+), is augmented by K+, but is diminished by Na+ and by ATP. Prior incubation with the aluminum chelator deferoxamine markedly slows inactivation, whereas adding 1 µM AlCl3 speeds it, consistent with AlF
–
4
being the active species. Prior incubation of the enzyme with vanadate also blocks inactivation by fluoride added subsequently. Fluoride stimulates ouabain binding to the enzyme, and thus the analogy between AlF
–
4
and both orthophosphate and orthovanadate is reflected not only in the similar dependence on specific ligands for their enzyme interactions and their apparent competition for the same sites, but also in their common ability to promote ouabain binding. Beryllium also irreversibly inhibits the enzyme, and this inactivation again requires divalent cations, is augmented by K+, but is diminished by Na+ and by ATP. Similarly, prior incubation of the enzyme with vanadate blocks inactivation by beryllium added subsequently. Inactivation by beryllium, however, does not require a halide, and, unlike inactivation by fluoride, increases at basic pHs. These observations suggest that beryllium, as beryllium hydroxide complexes, acts as a phosphate analog, similar to AlF
–
4
and vanadate.Abbreviations EDTA
ethylenediaminetetraacetate
- EGTA
Ethyleneglycol-bis(-aminoethylether)-N,N-tetraacetate
- FITC
fluorescein isothiocyanate
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid)1,4-piperazine diethanesulfonic acid 相似文献
15.
We demonstrate in this work that HCO
inf3
sup–
uptake in the marine macroalga Ulva sp. features functional resemblances to anion transport mediated by anion exchangers of mammalian cell membranes. The evidence is based on (i) competitive inhibition of photosynthesis by the classical red-blood-cell anion-exchange blockers 4,4-dinitrostilbene-2,2-disulfonate and 4-nitro-4-isothiocyanostilbene-2,2-disulfonate under conditions where HCO
inf3
sup–
, but not CO2, was the inorganic carbon form taken up; (ii) inhibition of HCO
inf3
–
uptake by pyridoxal phospate, indicating the involvement of lysine residues in the binding/translocation of HCO
inf3
sup–
; and (iii) inhibition of HCO
inf3
sup–
(but not of CO2) uptake by exofacial trypsin treatments, indicating the functional involvement of a plasmalemma protein. It is suggested that HCO
inf3
sup–
uptake mediated by such a putative anion transporter can be a fundamental step in providing inorganic carbon for the CO2-concentrating system of marine marcoalgae in an environment where the HCO
inf3
sup–
concentration is high, but the CO2 concentration and rates of uncatalyzed HCO
inf3
sup–
dehydration are low.Abbreviations CI
ionorganic carbon
- DIDS
4,4-diisothiocyanostilbene-2,2-disulfonate
- DNDS
4,4-dinitrostilbene-2,2-disulfonate
- NIDS
4-nitro-4-isothiocyanostilbene-2,2-disulfonate
- PLP
pyridoxal phosphate
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase 相似文献
16.
The reaction of ImpA in the presence of Na+-montmorillonite 22A or Na+-Volclay in aqueous, pH 8 solution gives a 50–60% yield of dimers and trimers (pA)2 and (pA)3. The ratio of 3,5-phosphodiester bond formation is twice as great as 2,5-bond formation. The reaction requires the presence of Mg2+ and is inhibited by 0.4 M imidazole. N-methylimidazole enhances the rate of the reaction but does not cause major changes in yield or product composition. Higher yields were obtained when Li+- or Ca2+-montmorillonites were used in place of Na+-montmorillonite. Little or no phosphodiester bond formation was observed with Mg2+- or Al3+-montmorillonite. Montmorillonites other than 22A and Volclay exhibited little or no catalysis. In addition, little or no catalysis was exhibited in ferrugenous smectite, nontronite, allophane, imogolite or sepiolite. Oligomers were also formed by the reaction of ImpG, 2-methylImpG, ImpC and ImpU in the presence of Na+-montmorillonite. The pyrimidine nucleotides gave significantly lower yields of oligomers. 相似文献
17.
Rajesh Mahey Michael A. Bridges Sidney Katz 《Molecular and cellular biochemistry》1991,105(2):137-147
Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca2+-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca2+-transporter showed high affinity for Ca2+ (KCa
2+ = 0.04 ± 0.01 M), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca2+-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5–1 mM), a high affinity Ca2+-dependent ATPase activity was observed (KCa
2+ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca2+-transport nor high affinity Ca2+-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg+)-ATPase activity, the presence of both a high affinity Ca2+-pump and high affinity Ca2+-dependent ATPase. However, the high affinity Ca2+-ATPase activity does not appear to be the biochemical expression of the Ca2+-pump.Abbreviations Ca2+-ATPase
calcium-activated, magnesium-dependent adenosine triphosphatase
- CaM
calmodulin
- CDTA
trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate
- EDTA
ethylene-diaminetetraacetate
- EGTA
ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate
- NADPH
reduced form of nicotinamide adenine dinucleotide phosphate 相似文献
18.
Hiroaki Sawai 《Journal of molecular evolution》1988,27(3):181-186
Summary Polymerization of various nucleoside-5-phosphorimidazolides has been conducted in neutral aqueous solution using divalent metal ions as catalysts. Oligonucleotide formation took place from each of the ribonucleoside-5-phosphorimidazolides, ImpC, ImpU, ImpA, ImpG, and ImpI. The yields and distributions of the resulting oligonucleotides varied depending on the difference of the nucleic acid base and the metal ions used. The catalytic effect of divalent metal ions on the formation of oligocytidylates occurred in the following order: Pb2+>Zn2+>Co2+, Mn2+>Cd2+>Cu2+>Ni2+>Ca2+, Mg2+, none >Hg2+. The order changes slightly for other types of oligoribonucleotide formation. Oligoribonucleotides up to hexamers were obtained in 35–55% overall yield, when Pb2+ ion was used as a catalyst. Zn2+ ions yielded oligoribonucleotides up to tetramers in 10–20% overall yield. The resulting oligonucleotides contained mainly 2–5 internucleotide linkages.Little or no oligonucleotide was obtained from nucleoside-5-phosphorimidazolides modified in the sugars, Imp(3-dA), Imp(2-dA), Imp(Ara), Imp(Aris), and Imp(Nep). The results indicate that a ribosyl system is required for the metal ion-catalyzed synthesis of oligonucleotides.
Abbreviations. EDTA, ethylenediaminetetraacetic acid; Versenol,N-hydroxyethylethylenediaminetriacetic acid; Tris, tris-(hydroxymethyl)aminomethane; pN (N is A, C, G, U, I, 3-dA, 2-dA, AraA, Aris, or Nep), nucleoside-5-phosphate; Np, nucleoside-2(3)-phosphate; I, inosine; 3-dA, 3-deoxyadenosine; 2-dA, 2-deoxyadenosine; AraA, arabinosyladenine; Aris, aristeromycin; Nep, neplanocin A; ImpN, nucleoside-5-phosphorimidazolide; NppN, P1,P2-dinucleoside-5,5-pyrophosphate; (pN)n (n=2, 3, ...), oligomers of pN, numbers given between a nucleoside and a phosphate indicate the type of internucleotide linkage, e.g., pC2 p5C is 5-phosphorylcytidyl-(2–5)-cytidine;
, cyclic dimers of pN; BAP, bacterial alkaline phosphatase; N.Pl, nuclease Pl; VPDase, venom phosphodiesterase; HPLC, high pressure liquid chromatography 相似文献
19.
Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts 总被引:1,自引:0,他引:1
An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg2+ up to 168% of the original activity, while Zn2+, Mn2+ and Cu2+ ions, chelating agent (EDTA) and NaN3 (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C. 相似文献
20.
Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E
0=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc
Gas liquid chromatography
- HPLC
high performance liquid chromatography
- RP
reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E
0 in mV)
- CAV2+
carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E
0=-296 mV)
- BV2+
benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E
0=-360 mV)
- MV
methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E
0=-444 mV)
- DMDQ2+
dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E
0=-514 mV)
- TMV2+
tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E
0=-550 mV)
- PDQ2+
propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E
0=-550 mV)
- DMPDQ2+
dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E
0=-656 mV)
- PN
productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)-1×h-1 相似文献