Fractionation of liver microsomes with polyethylene glycol and purification of NADH-cytochrome b5 oxidoreductase and cytochrome b5.
A simplified, rapid procedure for the purification of NADH-cytochrome b5 oxidoreductase and cytochrome b5 from either rat or rabbit liver is described. Microsomes were prepared by fractionation with polyethylene glycol and solubilized with Triton X-100. Cytochrome b5 was purified by a two-column procedure, anion exchange chromatography using DEAE-cellulose, and hydrophobic chromatography on phenyl-Sepharose. The final preparation of cytochrome b5 was purified more than a 120-fold from rat or rabbit liver microsomes, with specific content of about 50 nmol per mg protein, and overall yield of 22 to 32%. Only a single band with mol wt of 18,600 was found on sodium dodecyl sulfate (SDS)-gels or on Western blots using a polyclonal antibody raised against the purified b5. NADH-cytochrome b5 oxidoreductase was purified by a three-column procedure, DEAE-cellulose, hydroxylapatite, and ADP-agarose. The final product was purified more than 400-fold from rat or rabbit liver microsomes with a yield of about 25% and final specific activity of about 1600 mumol ferricyanide reduced per minute per milligram of protein. A single band with mol wt of 33, 100 was found on SDS-gels. The reductase catalyzed reduction of ferricyanide, dichlorophenol-indophenol, and cytochrome b5. Cytochrome c was reduced in the presence of reductase plus cytochrome b5, and this was inhibited by the anti-b5 IgG. The reductase catalyzed a rapid rate of reduction of ferric-ATP, which was slightly elevated by cytochrome b5. Ferric-histidine and ferric-ammonium sulfate were slowly reduced by reductase; addition of cytochrome b5 markedly stimulated reduction of these ferric complexes but inhibited reduction of ferric-EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)
Yang
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Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
Cederbaum
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D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D003579:Cytochrome Reductases / Q000302:isolation & purification*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000302:isolation & purification*; D005290:Ferric Compounds / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000737:chemistry*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols / Q000737:chemistry*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D005290:Ferric Compounds; D011092:Polyethylene Glycols; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase