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31.
Preparation of oligonucleotides corresponding to the acceptor stem of yeast tRNAPhe and their interaction with yeast ATP(CTP):tRNA nucleotidyltransferase 总被引:1,自引:1,他引:0 下载免费PDF全文
Seven oligonucleotides corresponding to the 3' and 5' sequences of the acceptor stem of yeast tRNAPhe have been prepared by chemical synthesis, chemical-enzymatic synthesis or by isolation from tRNA hydrolysates. The oligonucleotides have been examined as substrates for phosphodiester bond synthesis in the presence of ATP as catalysed by yeast ATP (CTP): tRNA nucleotidyltransferase. Oligonucleotides which correspond to the sequence of the 3'-strand of the tRNA acceptor stem and possess no secondary structure exhibit little or no activity with the enzyme. The ability of the enzyme to catalyse the synthesis of a phosphodiester linkage using ATP and an oligonucleotide corresponding to the 3'-strand of the acceptor stem is in general dramatically increased when an oligonucleotide corresponding to the sequence of the 5'-strand of tRNA acceptor stem is present. In cases where significant activity was observed kinetic parameters have been determined. 相似文献
32.
The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e, hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100 +/- 10 ms, compared to 6-10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a t 1/2 = 25-35 ms. The addition of uncouplers, which caused stimulatory effect on ferricyanide reduction under the same experimental conditions resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems. 相似文献
33.
Effects of vitamin K on human neutrophil function 总被引:1,自引:0,他引:1
J I Gallin B E Seligmann E B Cramer E Schiffmann M P Fletcher 《Journal of immunology (Baltimore, Md. : 1950)》1982,128(3):1399-1408
34.
Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated.It was found that:
- 1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide.
- 2) with or without cyanide in the incubation medium, LG omitted, Mn++, in the presence of NADPH induced superoxide anion (O¯2) production, as evidenced by oxygen consumption and H2O2 production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O¯2 scavenger).
- 3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occured only in the presence of Mn++, and both were inhibited by superoxide dismutase.
- 4) Cyanide-resistant NADPH oxidation by LG generated H2O2, was inhibited by H2O2 and was not modified by «active catalase. The ratio of cyanide-resistant NADPH oxidation/O2 uptake was 1 up to 1.25 mM NADPH, and increased above this concentration.
- 5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H2O2. The ratio of cyanide-sensitive NADPH oxidation/O2 uptake was 2.
35.
S-Alkylated 2-thiocytidine can be substituted at C-2 by nucleophilic agents. This reaction has been investigated with model compounds as well as with tRNA using the amino acids cysteine and lysine in order to develop a new affinity label linking covalently tRNA and a protein. Reaction with N-protected cysteine gives 2-S-alkyl-pyrimidines, while unprotected cysteine yields an N-alkyl-pyrimidine, after intramolecular substitution. With the -amino group of lysine a fast replacement at C-2 is observed, leading to an unstable 2-N-alkyl-pyrimidine. All products have been characterized both chemically and spectroscopically. 相似文献
36.
Summary About one third of the Dutch environmental research is concentrated on aquatic problems. The largest number of these projects is physicochemical (65%) and 47% of the aquatic projects contains an ecological component. The aquatic research projects are executed in a large number of different research institutes. Many institutes just formulated one project (43.2%) and about 10% of the institutes formulated 10 projects. The institutes with 10 or more projects account for almost 50% of the total number of projects. However, the size of the research projects with respect to the average total personnel per year may differ considerably. The largest number of aquatic research projects is carried out in governmental institutes. This relative high share of governmental institutes has proportionally increased during the period of 1975–1982. During this period the relative shares of all the aquatic research projects with an ecological component, of the strictly ecological projects and of the ecological/physicochemical projects have also proportionally inclined. However, in absolute numbers there seems to be a decline of both ecological and non-ecological projects on aquatic problems. 相似文献
37.
Iodometric measurement of lipid hydroperoxides in human plasma 总被引:4,自引:0,他引:4
Many assay techniques have been used to measure lipid hydroperoxides in plasma, including absorbance of conjugated dienes and reactivity with thiobarbituric acid. Because these measurements are not specific for lipid hydroperoxides, we modified an exisiting iodometric method to correct for interfering phenomena and to provide a more specific measurement of the lipid hydroperoxide content of plasma. To ensure reproducible extraction of hydroperoxides from the many possible forms in plasma, the plasma was treated to hydrolyze enzymatically cholesterol ester, triglycerides, and phospholipids, and the nonesterified fatty acid peroxides were then extracted with ethyl acetate. Extracted lipids were reacted with potassium iodide in acetic acid and methylene chloride, and the resulting triiodide ion (I3-) was measured spectrophotometrically. Correction for nonoxidizing chromophores was made after back-titration of the triiodide ion to iodide with sodium thiosulfate and other non-peroxide oxidants were estimated by their resistance to reduction with glutathione peroxidase. Recovery of added hydroperoxide standards provided routine validations of the procedure's efficiency. The method indicated that insignificant amounts of hydroperoxide may be in the less polar lipids, but the total amount of lipid hydroperoxide esterfied in the plasma lipids of apparently healthy humans may be as much as 4.0 +/- 1.7 microM. 相似文献
38.
On the nature of the structural change of the colicin E1 channel peptide necessary for its translocation-competent state 总被引:5,自引:0,他引:5
Acidic pH conditions required in vitro for membrane binding and activity of the channel-forming colicin E1 resulted in an increased susceptibility to proteases of the 178-residue thermolytic channel peptide, an increased accessibility to acrylamide of a fluorescence probe linked to cysteine-505 of the peptide, and an increased partition into nonionic detergent. The structural change in the peptide sensed by the fluorescence probe caused by a transition from pH 6.0 to 3.5 occurred in less than 1 s. The presence of low concentrations of detergents (0.001% SDS or 0.44% octyl beta-D-glucoside) or urea (0.2 M) at pH 6 or 4 also increased the susceptibility of the channel peptide to proteases. The increase in protease susceptibility and acrylamide accessibility at low pH, as well as partition of the peptide into nonionic detergent, suggested that acidic pH or the detergents might cause peptide unfolding. However, the hydrodynamic radius of the channel peptide at pH 6, 21-23 A, was not changed at pH 3.5 or by detergents or urea under conditions that increased the susceptibility of the peptide to protease. The activity of the channel peptide at pH 6 measured with liposomes and planar bilayers, which was a factor of 10(3)-10(4) smaller than that at pH 4, was increased by 2-4 orders of magnitude by 0.001% SDS or 0.44% octyl beta-D-glucoside, with an additional small increment of activity on planar bilayers caused by 0.01% SDS. A small increase in Stokes radius of the peptide in the presence of SDS could be detected that was approximately correlated with increased activity. 相似文献
39.
The assignments of the resonances of the methyl and methylene groups belonging to the residues dihydro-uridine-16 and -17 (C5 and C6), dimethylguanosine-26, N-2-methylguanosine-10, and 7-methylguanosine-46 of yeast tRNAPhe at low temperature are reported. Observing the high-field proton NMR spectral region at different temperatures, the effects of aminoacylation, removal of the Y base, and codon-anticodon interaction on the tertiary structure of yeast tRNAPhe were investigated. The following are the results of this study. (1) The two dihydrouridine residues of tRNAPhe have different environments in aqueous solution: dihydro-uridine-16 is more shielded than dihydrouridine-17. (2) The ribothymidine residue from the fragment (47--76) of yeast tRNAPhe and from a tRNA with a partially disrupted structure exhibits multiple conformations arising from different stacking modes between the ribothymidine-54 and the guanosine-53 residue. (3) Upon aminoacylation the type of guanosine-53 interaction with ribothymidine-54 in the tRNAPhe changes. (4) Removal of the Y base from the anticodon loop of yeast tRNAPhe weakens the thermal stability of the tertiary interactions. (5) The interaction of two complementary anticodons in the absence of proteins and of ribosomes results in stabilization of the tertiary structure. Codon-anticodon interaction dependent rearrangement of the tertiary structure of yeast tRNAPhe was not observed. The spin-lattice relaxation times of the methyl and methylene groups of the minor nucleosides in yeast tRNAPhe demonstrate that the minor nucleosides undergo rotational reorientation (tau c) in the nano-second range. The observed differences in these tau c values indicate a similarity of structure of tRNAPhe in solution and in crystalline form. 相似文献
40.
2-Thiocytidine 5'-triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase. s2CMP can be incorporated into both cytidine sites of the C-C-A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions. This was shown by alkylation of the 2-thiocytidine residues with iodo[14C]acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs. The incorporation of the 3'-terminal AMP is not influenced by the additional s2CMP residues at pH 9.0. However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position. Two different tRNAs with terminal 2-thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase. This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2-thiocytidine in the normal position 75. The addition of 2-mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA. This reaction identifies the attacking nucleophile of the enzyme as cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously. The mechanism of the enzymatic and chemical reactions is discussed. 相似文献