NADPH-cytochrome P-450 reductase-cytochrome b5 interactions: crosslinking of the phospholipid vesicle-associated proteins by a water-soluble carbodiimide.
Detergent-solubilized and purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome b5 were coreconstituted into phospholipid vesicles. When the proteoliposomes were incubated with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a new higher-molecular-weight band was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The band was purified by chromatography on DEAE-Sepharose CL-6B, 2'5'-ADP-Sepharose 4B, and Sephadex G-100. The heme absorption spectrum and fluorophotometric assay of flavin of the purified material demonstrate that this product is a 1:1 crosslinked complex containing one molecule each of the flavoprotein and cytochrome. Proteolysis of the crosslinked form indicates that the hydrophilic catalytic domains participate in the covalent attachment, and that the hydrophobic membrane-attachment peptide is necessary for the protein interaction. The purified crosslinked derivative showed no activities for reduction of either cytochrome c or ferricyanide. About half of the enzyme-associated flavin was reduced rapidly by NADPH, as was 20-30% of the crosslinked cytochrome, indicating that, in at least some of the complexes, the flavin-mediated pathway for reduction of cytochrome by pyridine nucleotide was intact. These data suggest that the output- rather than the input-electron transfer site(s) in the flavoprotein was (were) blocked by the covalently attached cytochrome.
D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism*; D015786:Cytochromes b5; D003840:Deoxycholic Acid / Q000494:pharmacology; D004579:Electron Transport; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism*; D011817:Rabbits; D012995:Solubility
D002234:Carbodiimides; D002352:Carrier Proteins; D003573:Cytochrome b Group; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D003840:Deoxycholic Acid; D009249:NADP; D015786:Cytochromes b5; D009251:NADPH-Ferrihemoprotein Reductase; D005022:Ethyldimethylaminopropyl Carbodiimide