Interaction of non-myristoylated NADH-cytochrome b5 reductase with cytochrome b5-dimyristoylphosphatidylcholine vesicles.
An expression vector for NADH-cytochrome b5 reductase containing a thrombin cleavage site directly before the N-terminal glycine residue of the flavoprotein was used to isolate the non-myristoylated enzyme by thrombin cleavage of the initial fusion protein of a short segment of the multiple cloning site of the plasmid vector and the reductase. This flavoprotein preparation, containing only the 28-residue N-terminal peptide segment of the membrane-binding domain of the mammalian enzyme, binds to phospholipid vesicles and interacts with membrane-bound cytochrome b5. The effect of N-myristoylation of the enzyme therefore appears to be limited to facilitating and stabilizing interactions with phospholipid vesicles. However, the relatively short intervening peptide sequence that separates the crucial peptide membrane-binding domain from lysine 41, which has been implicated in the active-site interaction with cytochrome b5 (Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) J. Biol. Chem. 267, 2519-2523), provides some limitation of the distance from the membrane surface for the interactions required for rapid electron transfer from the flavin of the reductase to the heme of cytochrome b5.
Strittmatter
P
P
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030-3305.
Kittler
J M
JM
Coghill
J E
JE
Ozols
J
J
D001483:Base Sequence; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000378:metabolism; D008969:Molecular Sequence Data; D009226:Myristates / Q000378:metabolism*; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D017931:DNA Primers; D008563:Membrane Lipids; D009226:Myristates; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D004134:Dimyristoylphosphatidylcholine