TY - JOUR
T1 - Fractionation of liver microsomes with polyethylene glycol and purification of NADH-cytochrome b5 oxidoreductase and cytochrome b5
AU - Yang, Ming Xue
AU - Cederbaum, Arthur I.
PY - 1994/12
Y1 - 1994/12
N2 - 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 microsemes, 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 μmol 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. The procedures described result in highly purified preparations of reductase and cytochrome b5 with relatively good yield and require fewer chromatographic stops than previously reported.
AB - 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 microsemes, 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 μmol 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. The procedures described result in highly purified preparations of reductase and cytochrome b5 with relatively good yield and require fewer chromatographic stops than previously reported.
KW - Cytochrome b
KW - NADH-cytochrome b reductase
KW - Polyethylene glycol
KW - Purification
UR - http://www.scopus.com/inward/record.url?scp=0027973889&partnerID=8YFLogxK
U2 - 10.1006/abbi.1994.1522
DO - 10.1006/abbi.1994.1522
M3 - Article
AN - SCOPUS:0027973889
SN - 0003-9861
VL - 315
SP - 438
EP - 444
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -