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1.
The reaction of adenine and cytosine residues in tRNA with chloroacetaldehyde 总被引:1,自引:1,他引:0
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W J Krzyzosiak J Biernat J Ciesio?ka K Gulewicz M Wiewiórowski 《Nucleic acids research》1981,9(12):2841-2851
The reaction of Lupinus luteus tRNAPhe with 1 M chloroacetaldehyde in the pH range of 4 - 6 at 25 degrees C was studied. It was found that earlier difficulties lowering the utility of the reagent for structural studies of nucleic acids were caused by the formation of the stable reaction intermediates. In order to eliminate these difficulties the simple procedure of so-called maturation of the chloroacetaldehyde-modifies tRNA is proposed. It consists in further incubation of the short-term modified tRNA in water at 50 degrees C in the absence of the reagent. During the maturation step the stable intermediates are quantitatively converted into final ethenoderivatives. New HPLC conditions on Aminex A-6 were worked out which enable rapid separation of both ethenoadenosine and ethenocytidine from natural tRNA nucleosides. 相似文献
2.
Augusto Cesar C. Spadaro Antonio R. Martins Jane D. Berti Lewis J. Greene 《Analytical biochemistry》1978,91(2):410-420
An analytical method utilizing an automatic amino acid analyzer is described for the separation, identification, and measurement of 5 to 50 nmol of angiotensin I, angiotensin II, [Des-Phe8]angiotensin II, Phe-His-Leu, His-Leu, isoleucine, leucine, tyrosine, and phenylalanine. Aminex A-5 cation-exchange resin (0.9 × 15 cm) is sequentially eluted with three sodium citrate buffers: pH 3.25, 0.2 n; pH 4.85, 0.54 n, and pH 6.5, 0.39 n at 60 and 80°C. Reaction with ninhydrin is used for detection. This chromatographic system was used to determine angiotensin-converting enzyme activity and the angiotensinase activity of rabbit brain endopeptidase B. In each assay, the unhydrolyzed substrate and both products were measured simultaneously in one step without pretreatment of the hydrolysate. Products were recovered in 1:1 molar ratios and the overall recovery of unhydrolyzed substrate of products was quantitative. 相似文献
3.
J. Süss 《Biologia Plantarum》1972,14(6):385-389
Studying the influence of the pH of 10% NaCl solutions used for the extraction of RNA and DNA on the yield of both nucleic acids, the maxima of pH were found at which both types of nucleic acids pass into extracts better than at neutral pH and do not remain in residues of the experimental material.The perchloric acid extraction temperature was also studied for obtaining the hydrolysate of nucleic acids from the trichloroacetic acid precipitate of sodium chloride extracts differing by 5 °C within the range of 35 °C to 90 °C and it was found that in this wide range almost the same amount of RNA is extracted by the method used. However, at a lower temperature, some DNA remained in the extracted residue of the trichloroacetic acid precipitate of sodium chloride extracts. 相似文献
4.
tRNA2Leu from cow mammary gland has been degraded with pancreatic ribonuclease, and the fragments obtained were separated by DEAE-cellulose micro-column chromatography in 7 M urea at pH 7,5. Rechromatography was performed on a DEAE-cellulose micro-column at pH 3,7 and also on Dowex 1 X 2 in a formiate system. Nucleotide analysis was carried out with the aid of T2-RNase hydrolysis followed by chromatography on anion-exchanger AG 1 X 8. Nucleosides were separated on Aminex A-6 at pH 9,8. 15 minor components were shown to be present: T, 2 psi, 2Um, 2D, m5C, ac4C, m1G, 2m2G, m22G, m1A and N, the N is not identified so far. The structure of oligonucleotides was established by terminal analysis, hydrolysis with T1-RNase and also using incomplete hydrolysis by the snake venom phosphodiesterase. 相似文献
5.
Dinshaw J. Patel 《Biopolymers》1977,16(12):2739-2754
The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site. 相似文献
6.
Computational investigation of proton transfer,pKa shifts and pH‐optimum of protein–DNA and protein–RNA complexes
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Protein–nucleic acid interactions play a crucial role in many biological processes. This work investigates the changes of pKa values and protonation states of ionizable groups (including nucleic acid bases) that may occur at protein–nucleic acid binding. Taking advantage of the recently developed pKa calculation tool DelphiPka, we utilize the large protein–nucleic acid interaction database (NPIDB database) to model pKa shifts caused by binding. It has been found that the protein's interfacial basic residues experience favorable electrostatic interactions while the protein acidic residues undergo proton uptake to reduce the energy cost upon the binding. This is in contrast with observations made for protein–protein complexes. In terms of DNA/RNA, both base groups and phosphate groups of nucleotides are found to participate in binding. Some DNA/RNA bases undergo pKa shifts at complex formation, with the binding process tending to suppress charged states of nucleic acid bases. In addition, a weak correlation is found between the pH‐optimum of protein–DNA/RNA binding free energy and the pH‐optimum of protein folding free energy. Overall, the pH‐dependence of protein–nucleic acid binding is not predicted to be as significant as that of protein–protein association. Proteins 2017; 85:282–295. © 2016 Wiley Periodicals, Inc. 相似文献
7.
By using column chromatography on varied media, the purification of several individual tRNAs from human placenta has been achieved. The crude human placenta tRNA was isolated using phenol extraction at pH 4.5 followed by DEAE-cellulose chromatography (B. Roe (1975), Nucleic Acids Res. 2, 21-42) and initially fractionated on BD-cellulose at neutral pH. Subsequent chromatography of the partially purified tRNA using high-speed, high-pressure liquid chromatography on RPC-5 and Aminex A-28 coupled with chromatography on BD-cellulose at acidic pH and on DEAE-Sephadex A-50 significantly shortened isolation time for milligram quantities of several pure tRNA species. Those tRNAs from human placenta obtained in a purity greater than 1.2 nmol/A260 unit are tRNAPhe, tRNAMet(i), tRNAVal(1a), tRNAVal(1b), and tRNAGly(1), while those obtained at purity of at least 0.8 nmol/A260 unit are tRNASer2 and tRNASer3. In addition, the use of Aminex A-28 as a chromatographic system for the isolation of tRNA is discussed. 相似文献
8.
A psychrotolerant phosphate solubilizing fungus has been isolated from the rock soil of a cold desert site in Indian Himalaya.
The fungus grows from 4 to 35°C (optimum 21°C), and from 2 to 13.5 pH (optimum 9) under laboratory conditions. Based on phenotypic
characters and 26S rDNA analysis, the fungus is identified as Paecilomyces hepiali. In quantitative estimation that was carried out at 9, 14, and 21°C, the fungus solubilized maximum phosphate at 14°C. In
view of the slow growth and persistence of the desired activity at low temperature, the estimation was carried out for a longer
period, i.e., up to 6 weeks. The suboptimal conditions for growth and biomass production were found to be optimal for phosphate
solubilization by the fungus. At 14 and 9°C, the solubilization touched its maximum on day 42. Decline in pH was found to
be significantly correlated with the phosphate solubilization at all the temperatures, under consideration. The acid phosphatase
activity was found to be more prominent than alkaline phosphatase in culture filtrate. High performance thin layer chromatography
(HPTLC) analysis showed production of six organic acids, gluconic and α-keto glutaric acid being in maximum amount in the
culture filtrate. The study has ecological significance in view of the nutrient cycling under low temperature environment,
prevalent in Himalayan region. 相似文献
9.
A rapid and sensitive assay for individual uronic acids has been developed based on their separation on a 0.5 × 22-cm column of Aminex A-25 in 0.12 m Tris-acetate buffer, pH 7.4. Quantification of these sugars is accomplished by coupling the column to the analytical portion of a Technicon sugar analyzer. Each determination is complete in 3 hr, and as little as 25 nmol of uronic acid can be measured with accuracy. 相似文献
10.
Using the interaction of polyacrylic anhydride with uridine or N-acetyl derivatives of adenosine, cytidine, and guanosine, the water-soluble copolymers, polyacryloylnucleosides, were obtained. The acryloylnucleoside units to acrylic acid units ratio in the copolymer was usually about 1:20. The attaching of nucleosides to the polymer occurs mostly through the 5′-hydroxyl group of the sugar. The prominent feature of all polyacryloylnucleosides obtained is their fibrous structure at an ionic strength 0.2 and neutral pH. At concentrations <200 μg/ml the separate strands with a length of 0.2–1.0 μ and diameter of 30–40 Å are distinguishable. Evidently they are formed by side association of two molecules of copolymer or by folding of one molecule on itself. At higher concentrations branched multistranded structures are formed. In the same conditions polyacrylic acid alone does not form the fibrous structures. Heating of polyacryloylnucleoside solutions at 100°C and fast cooling in ice water, or raising of the pH to 12 turned the stranded structures to coils. After annealing or neutralization the stranded structures reformed. These transformations are similar to those which occur with nucleic acids. The results show that the fibrous structure of the copolymers depends on the hydrogen bonds formed by purine and/or pyrimidine bases. 相似文献
11.
Yoshiki Kumada Tomio Takeuchi Hamao Umezawa 《Bioscience, biotechnology, and biochemistry》2013,77(5):869-876
A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39,000. The optimal pH for the enzymic conversion of caffeic acid to dehydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10 min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C. 相似文献
12.
Bernhard Brosius Detlev Riesner Wolfgang Hillen 《Journal of biomolecular structure & dynamics》2013,31(6):1535-1541
Abstract The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg- lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5° to 74°C, the transition is broadened from 0.7°C to 2.7°C. Varying the phosphatidyl-serine-lecithin-ratio from 0–100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5°C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed. At present, the biological relevance of the results may be more in the drastic decrease in cooperativity than in the slight modulation of the stability. From nearly 180 base pairs opening up cooperatively in free nucleic acid this number is lowered to less than 50, a size in the range of promotor regions. 相似文献
13.
Nobumaro Kawashima Harui Fukushima Ayako Imai Einosuke Tamaki 《Bioscience, biotechnology, and biochemistry》2013,77(9):1141-1145
A leaf protease of tobacco whose activity was enhanced during curing was purified about 60 times with ammonium sulfate fractionation, ethanol precipitation, calcium phosphate gel treatment and Sephadex G-200 column chromatography, and some properties of the protease were examined. The purified enzyme showed the optimum pH at 5.5 and the optimum temperature at 60°C. The protease activity was stable between pH 4.5 and 5.5 at 50°G or at pH 5.5 below 40°C for 1 hr, but completely destroyed at 70°C during 1 hr. The protease activity was greatly activated by reducing agents such as cysteine, glutathione or mercaptoethanol and inhibited by p-chloromercuribenzoate, phenyl- mercuric acetate or silver ions. Metal ions except for silver ion and ethylenediamine tetraacetic acid did not affect the protease activity so far examined. 相似文献
14.
A phosphate solubilizing and antagonistic bacterial strain, isolated from a Western Ghat forest soil in Kerala province, India
(designated as NII-0906), showed cold tolerance and grew from 10 to 37°C (optimum temperature 30°C). It was a Gram-positive,
rod shaped, 0.8–1.6 μm in size, and exhibited tolerance to a wide pH range (5–12; optimum 7.0) and salt concentration up to
7% (w/v). The isolate showed maximum similarity with Exiguobacterium marinum TF-80T based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was
estimated along a temperature range (5–40°C), and maximum activity (84.7 μg mL−1 day−1) was recorded at 30°C after 10 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease
in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and
produced siderophore and hydrogen cyanide. The strain’s plant growth promotion properties were demonstrated through a cowpea-based
bioassay under greenhouse conditions. The bacterial inoculation resulted in significant increment in plant root, stem and
as well as in plant biomass. Further, scanning electron microscopic study revealed the root colonization in cowpea. These
results could offer potential perspective for the strain to be used as plant growth-promoting rhizobacteria, which could be
used as an inoculant for regional crops. 相似文献
15.
Purine nucleoside phosphorylase from Enterobacter cloacae KY3074 was partially purified by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and DEAE-Sephadex A-50, and gel filtration on Sephadex G-100 and Sepharose 4B. The molecular weight of the enzyme was calculated to be about 87,000 by a gel filtration method on Sephadex G-200. The enzyme was found to be most active at pH 7.5 to 8.5 and 50°C, stable between pH 7.0 and 7.3, and the activity was nearly lost above 70°C. The enzyme split 2´-deoxyinosine and ribonucleosides. Lineweaver-Burk plots for phosphate were non-linear, showing substrate activation. The break-down of inosine approached an equilibrium when approximately 14% of inosine was phosphorylated. 相似文献
16.
Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH2PO4 in 0.2 mM CaSCO4 at the average rate of 34.3 ±3.2 μg Pi g?1 (fr. m.) h?1,i.e. 0.35± 0.02 μmol Pi g?1 (fr. m.) h?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO4. This increase is completely blocked by the presence of 10 μg ml?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following Km and Vmax values were found: Km[mM] : 0.37 - 3.82 - 27.67 Vmax[μg Pi g?1 (fr. m.) h?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH2PO4, while uptake from 5 and 10 mM KH2PO4 was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10?3 M NEM by 81.6%, 104 M NEM by 42% and 10?4 M PCMB by 42%. 相似文献
17.
Gen-ichi Danno 《Bioscience, biotechnology, and biochemistry》2013,77(2):264-273
An alkaline proteinase of Aspergillus sulphureus (Fresenius) Thorn et Church has been purified in good yields from wheat bran culture by fractionation with ammonium sulfate, treatment with acrynol, and DEAE-Sephadex A-50 column chromatography. The crystalline preparation was homogeneous on sedimentation analysis and polyacrylamide gel zone electrophoresis. The molecular weight was calculated to be 23,000 by gel filtration. The amino acid composition of the enzyme was determined. The enzyme did not precipitate with acrynol. Optimum pH for the hydrolysis of casein was 7 to 10 at 35°G for 15 min. Optimum temperature was 50°C at pH 7 for 10 min. The enzyme was highly stable at the range of pH 6 to 11 at 5°C, whereas relatively stable at pH 6 to 7 at 35°C. Metalic salts tested did not affect activity. Chelating agents, sulfhydryl reagents, TPCK, and oxidizing or reducing reagents tested, except iodine, had no effect on the activity. Diisopro-pylfluorophosphate and N-bromosuccinimide almost completely inactivated the proteinase. 相似文献
18.
The crude lipase powder has been purified 216-fold in specific activity by means of pH adjustment, DEAE-Cellu1ose, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 column chromatography and the recovery of the activity was 30%. The purified lipase was confirmed to be homogeneous with disc electrophoresis and ultracentrifugal analyses. The purified lipase was stable in the pH range from 7.0 to 10.0. Optimal pH for the lipolysis of polyvinyl alcohol-emulsified olive oil at 45°C was 8.0 and optimal temperature was 60°C. The purified lipase was stable up to 60°C and retained 55% of full activity after heating at 70°C for 20 min. 相似文献
19.
Kei Arima Takeshi Uozumi Masato Takahashi 《Bioscience, biotechnology, and biochemistry》2013,77(11):1033-1050
The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases. 相似文献
20.
Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding Ki values ranging from 10.5 to 14.1 × 10?6 M in comparison to the corresponding apparent KM (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO4??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO4?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10?4 M), and adenosine triphosphate (1–5 × 10?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca++ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca++ and Mg++ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′2 = 6.14 and for pK′3 = 10.99, at 15°C and phosphate at infinite dilution. 相似文献