TAILIEUCHUNG - Báo cáo khoa học: Three mammalian cytochromesb561are ascorbate-dependent ferrireductases

Cytochromesb561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cyto-chromes b561 (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochromeb) were expressed in a Saccharomyces cerevisiae Dfre1Dfre2 mutant, which lacks almost all of its plasma membrane ferrire-ductase activity, to study their ability to reduce ferric iron (Fe 3+ ). | ễFEBS Journal Three mammalian cytochromes b561 are ascorbate-dependent ferrireductases Dan Su and Han Asard Department of Biochemistry University of Nebraska-Lincoln Lincoln NE USA Keywords ascorbate cytochrome b561 ferrireductase Correspondence H. Asard Department of Biology University of Antwerp Groenenborgerlaan 171 B-2020 Belgium Fax 32 3265 3417 Tel 32 3265 3638 E-mail Received 31 March 2006 revised 25 May 2006 accepted 13 June 2006 doi Cytochromes b561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b561 chromaffin granule cytochrome b duodenal cytochrome b and lysosomal cytochrome b were expressed in a Saccharomyces cerevisiae Dfre1Dfre2 mutant which lacks almost all of its plasma membrane ferrire-ductase activity to study their ability to reduce ferric iron Fe3 . The expression of each of these cytochromes b561 was able to rescue the growth defect of the Dfre1Dfre2 mutant cells in iron-deficient conditions suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b561 showed significant FeCN and Fe3 -EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site-directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated mutations of four His residues H47 H83 H117 and H156 one Tyr Y66 and one Arg R67 completely abrogated the FeCN reductase activity whereas mutations of Arg R149 Phe F44 Ser S115 Trp W119 Glu E196 and Gln Q131 affected the ferrireductase activity to some degree. These mutations may affect the heme coordination ascorbate binding and or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This .

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