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We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.  相似文献
3.
A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.  相似文献
4.
A new assay method for lipid peroxides using a methylene blue derivative   总被引:6,自引:0,他引:6  
To determine the absolute amount of lipid hydroperoxides in biological materials, a simple and sensitive colorimetric method was newly developed, based on the reaction of lipid hydroperoxides with a leucomethylene blue derivative in the presence of hemoglobin. The amount of methylene blue formed was measured by its absorbance at 666 nm to calculate the amount of lipid hydroperoxides using cumene hydroperoxide as external standard. By this method, lipid hydroperoxide concentrations of less than 7.5 nmol/tube were accurately determined.  相似文献
5.
We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.  相似文献
6.
The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.  相似文献
7.
The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [( Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76-79% identity at the amino acid level to the corresponding maize P450 species CYP71C1-C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast ( Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6- C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (approximately SS)] and Ae. squarrosa (DD).  相似文献
8.
Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130- and 135-kilodalton insecticidal proteins, respectively, of Bacillus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37 degrees C, but bipyramidal crystals practically free of contaminants were observed at 30 degrees C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.  相似文献
9.
The inhibitory effects of vitamins A and K toward P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined in the reconstituted system containing the microsomal fraction prepared from the recombinant Saccharomyces cerevisiae cells producing rat P4501A1 and yeast NADPH-P450 reductase. On vitamins A, all-trans-retinol, all-trans-retinal, all-trans-retinoic acid and retinol-palmitate showed competitive inhibition with K(i) values of 0.068, 0.079, 2.6 and 2.0 microM, respectively. Judging from the K(i) values, the inhibitory effects of those vitamins A appear to have physiological significance on the basis of their contents in liver, lung and kidney. On vitamins K, vitamin K(1) showed competitive inhibition with K(i) value of 24 microM, while vitamin K(2) showed noncompetitive inhibition with K(i) value of 60 microM. Judging from these K(i) values together with the contents of these vitamins K in liver, the inhibitory effects of the vitamins K are not as significant as those of vitamins A. These results suggest that the ingestion of enough amounts of vitamins A from foods might lead to the inhibition of the activity of P4501A1 which is known to be induced by smoking, drugs such as omeprazole and lansoprazole, and environmental pollutants like dioxins.  相似文献
10.
Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pGFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNG01 under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase gene terminator. The fused enzyme cDNA was integrated into tobacco genomes by Agrobacterium infection techniques. In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction. The microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo[a]pyrene than in the control plant. The transgenic plants also showed resistance to the herbicide chlortoluron.  相似文献
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