Binding of bovine cytochrome b5 to phosphatidycholine liposomes Characterization of the reconstituted lipid-protein vesicles

Dufourcq, Jean; Bernon, R.; Lussan, Claude

doi:10.1016/0005-2736(76)90091-2

Cited by 37 publications

(2 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…First, where the procedure calls for gel chromatography with Sephadex G-75, we use Sephadex G-150 instead, since we find that this modification more effectively removes the brownish high molecular weight impurities. In the purified preparations, the ratio of the absorbance at 413 nm to that at 280 nm is 2.7, which is equal to the best values reported in the literature (Dufourcq et al, 1976;Spatz & Strittmatter, 1971). Only one band is visible on SDS-polyacrylamide slab gels when 1 ug of protein is applied, but if the gel is overloaded with 10-15 µg of protein, several faint bands at higher molecular weight are visible.…”

Section: Methodssupporting

confidence: 66%

See 1 more Smart Citation

Cytochrome b5 induced flip-flop of phospholipids in sonicated vesicles

Greenhut¹,

Roseman²

1985

Biochemistry

View full text Add to dashboard Cite

Cytochrome b5 induced flip-flop of phosphatidylethanolamine (PE) in sonicated vesicles prepared from a 9:1 mixture of phosphatidylcholine (PC) to phosphatidylethanolamine was determined as follows. First, vesicles having a nonequilibrium distribution of PE across the bilayer were prepared by amidinating the external amino groups with isethionyl acetimidate. Amidinated cytochrome b5 was then added, and after the protein was completely bound, the rate of appearance of fresh PE on the outer surface was determined by removing aliquots at timed intervals and titrating the external amino groups with trinitrobenzenesulfonic acid. The results show an initial rapid phase of flip-flop (especially in the presence of salt) followed by a very slow phase, at 25 degrees C. Similar results were obtained when cytochrome b5 was introduced into the amidinated vesicles by spontaneous transfer from PC donor vesicles. These results indicate that the accumulation of the transferable ("loose") form of cytochrome b5 on the outer surface of a vesicle causes a transient, global destabilization of the bilayer that is relieved by lipid flip-flop. We speculate that this mechanism may be a significant driving force for the transfer of amphipathic molecules across membranes.

show abstract

Section: Methodssupporting

confidence: 66%

“…Second, we always run the final Sephadex G-25 gel filtration step twice to ensure complete removal of detergent. Purification with tritiated DOC has shown that the residual DOC is only 1 mol per 100 mol of cytochrome b¡ (Dufourcq et al, 1976).…”

Section: Methodsmentioning

confidence: 99%