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FUKUTOME has once reported that isovaleric acid is an isovalthinuria inducer but isovaleric acid-1-C14 administered to a dog does not incorporate into urinary isovalthine and glutamic acid is most strongly labeled among acidic amino acids excreted. Recently, however, KUWAKI has found that liver homogenates of some animals can synthesize C14-labeled S-(isopropylcarboxymethyl) glutathione (GSIV) from isovaleric acid-1-C14 and glutathione, and that GSIV can be converted into isovalthine by kidney homogenate or glutathionase preparation4. For the elucidation of the above discrepancy, FUKUTOME's experiments were repeated by using isovaleric acid-methyl-C14 or-1-C14, and it was again found that these isotopic compounds did not significantly incorporate into urinary isovalthine.

Cytochrome P-450 is involved in the oxidative metabolism of a broad range of substrates. We have made a chimeric construct, pSN002, containing the cDNA for rabbit liver cytochrome P-450 (IIC14) under the control of the TR2' promoter for mannopine synthase in the Agrobacterium Ti plasmid. Nicotiana tabacum was transformed with Agrobacterium tumefaciens harboring a cointegrated plasmid pSN002::pGV2260. The presence of mRNA and of the translated protein from the chimeric cytochrome P-450 gene in transgenic plants was confirmed by RNA blot hybridization and by Western blot and immunohistochemical analyses, respectively. The transformants in which the foreign cytochrome P-450 protein is expressed show marked phenotypic changes, notably a tendency rapidly to senesce. We detected 2-propenylpyrrolidine, a degradative metabolite of nicotine alk...
Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for th...
Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 (P450) and other enzymes involved in the biotransformation of drugs and other xenobiotics. The list of studies made possible with the technology includes discernment of catalytic specificity, elucidation of structure-activity relationships, and various biophysical measurements. There are advantages and disadvantages to each of the vector systems and choices must be made on the basis of needs. Yeast expression systems were used to establish that different P450 2C enzymes are involved in the hydroxylations of tolbutamide and (S)-mephenytion. P450 3A4 was also expressed in yeast and its very broad catalytic specificity was confirmed. Recently, it has been possible to express P450 3A4 as well as other human and animal P450s in bacteria ...
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