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71.
Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole. 相似文献
72.
足月分娩的新鲜胎盘组织制成匀浆后,经高速离心、超速离心,谷胱甘肽(GSH)Sepharose 6B亲合层析,Amicon pM-10膜超过滤及高效液相层析,最终经SDS-PAGE鉴定,结果呈现单一亚基区带,其亚基分子量为25000。 根据我们现有高效液相设备条件,用ODS柱代替RadulovicL等报道的特异阴离子柱,用磷酸盐洗脱液代替含谷胱甘肽、二硫苏糖醇及氯化钾的梯度洗脱液,从人胎盘组织成功地制备了谷胱甘肽硫转移酶(GST)纯酶,全过程在15min内完成,保留时间及主峰面积的重复性均较理想,7次实验结果的变异系数为0.2%,最终纯化578.9倍。本研究为各种形式GST的纯化制备提供了一个新的、重复性好、分辨率高及回收理想的简易方法。 相似文献
73.
Adenine occurs in the strand containing repeated G clusters in the telomeric DNA of a variety of organisms, including that of humans. The role of adenine has been investigated by constructing two sets of oligonucleotides each with one, two, or four copies of the telomeric sequence dTTTAGGG together with a control sequence in which T replaces the A residue, dTTTTGGG. Comparison of the stability and spectral properties of these two sequences in the presence of Na+ or K+ affords a basis for defining the role of adenine in these structures. In Na+, the A residue stabilizes the structure formed by each oligomer significantly, presumably by a base-pairing interaction with T. In K+, by contrast, there is little difference in stability. In two- and four-copy oligomers, the A sequence has a different structure from its T analog, as detected by CD spectroscopy. In the presence of either Na+ or K+, the tetraplexes of A and T interact with intercalators. 相似文献
74.
Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor. 相似文献
75.
Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence. 相似文献
76.
A large body of in vitro and in vivo data suggests that combinations of cytokines provide the most effective mechanism for stimulating multilineage acceleration of hematopoiesis. Creation of a granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein has yielded a single therapeutic which has enhanced biological activity in comparison to the individual cytokines from which it is composed. In vivo studies with this fusion protein (PIXY321) suggest that it may provide a means to accelerate both neutrophil and platelet recovery in clinical settings in which hematopoiesis is suppressed. The biology of PIXY321 and the potential for other fusion proteins is discussed. 相似文献
77.
An immunodetection assay on Western blots has been used to determine the thiol proteinase content and composition in cysts from 12 populations of the brine shrimp Artemia. Our results showed no differences in the subunit composition of the thiol proteinase among cysts from eight bisexual strains and four parthenogenic strains, and confirmed an earlier finding that the proteinase is composed of two subunits of 25.9 and 31.5 kilodaltons. In contrast, we found that Artemia cysts from parthenogenic strains contain 17.1 ng/cyst of the thiol proteinase, while cysts from bisexual strains contain 8.2 ng/cyst of the thiol proteinase. Also, there was a good linear correlation (r = 0.863; p less than 0.001) between the thiol proteinase content and cyst mass. Embryo fractionation experiments showed that 82% of the thiol proteinase was in the cytosol, while 14 and 4%, respectively, were in the nuclei/yolk platelets and mitochondria/lysosome fractions. Measurements of the thiol proteinase content of developing Artemia embryos showed that the proteinase content was relatively constant during early development, suggesting that the activity of the thiol proteinase gene(s) may be constitutive and not developmentally regulated in Artemia embryos. 相似文献
78.
Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems. 总被引:22,自引:9,他引:13 下载免费PDF全文
Nitrogen control in Salmonella typhimurium is not limited to glutamine synthetase but affects, in addition, transport systems for histidine, glutamine, lysine-arginine-ornithine, and glutamate-aspartate. Synthesis of both glutamine synthetase and transport proteins is elevated by limitation of nitrogen in the growth medium or as a result of nitrogen (N)-regulatory mutations. Increases in the amounts of these proteins were demonstrated by direct measurements of their activities, by immunological techniques, and by visual inspection of cell fractions after gel electrophoresis. The N-regulatory mutations are closely linked on the chromosome to the structural gene for glutamine synthetase, glnA: we discuss the possibility that they lie in a regulatory gene, glnR, which is distinct from glnA. Increases in amino acid transport in N-regulatory mutant strains were indicated by increased activity in direct transport assays, improved growth on substrates of the transport systems, and increased sensitivity to inhibitory analogs that are trnasported by these systems. Mutations to loss of function of individual transport components (hisJ, hisP, glnH, argT) were introduced into N-regulatory mutant strains to determine the roles of these components in the phenotype and transport behavior of the strains. The structural gene for the periplasmic glutamine-binding protein, glnH, was identified, as was a gene argT that probably encodes the structure of the lysine-arginine-ornithine-binding protein. Genes encoding the structures of the histidine- and glutamine-binding proteins are not linked to glnA or to each other by P22-mediated transduction; thus, nitrogen control is exerted on several unlinked genes. 相似文献
79.
The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity. 相似文献
80.