Study of lipid—protein interactions in membrane models: Intrinsic fluorescence of cytochrome <i>b</i><sub>5</sub>—phospholipid complexes

Dufourcq, Jean; Faucon, Jean‐François; Lussan, Claude; Bernon, R.

doi:10.1016/0014-5793(75)80164-5

Cited by 43 publications

(16 citation statements)

References 25 publications

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“…The highest D-,/-hydroxybutyrate dehydrogenase fluorescence intensity increase obtained in the presence of mitochondrial phospholipid confirms the crucial role of phosphatidylcholine in the correct insertion of this enzyme, its conformation and, consequently, its biological function. The effect of phospholipids on the fluorescence intensity of tryptophan residues observed without shift of maximum emission wavelength has been previously obtained with cytochrome b5 [16] and with apolipoprotein GL N1 [29]. By using the fluorescence life-time technique (results not shown), we have observed that the fluorescence life-time of tryptophan residues of the enzyme increases significantly after insertion of the enzyme into phosphatidylcholine vesicles, whereas it remains unchanged in the enzyme reconstituted with phosphatidylethanolamine/diphosphatidylglycerol micro-dispersions [30].…”

Section: Discussionmentioning

confidence: 88%

“…Fluorescence is a very powerful technique used in the investigation of protein motions in a defined microenvironment and in the study oflipid-protein interactions [16][17][18][19]. In order to obtain more information concerning the role of phospholipids in D-fl-hydroxybutyrate dehydrogenase structure and in its insertion into liposomes, we report a fluorescence study of the interaction of the purified rat liver enzyme with phospholipids.…”

Section: Introductionmentioning

confidence: 99%

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Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

Kebbaj¹,

Latruffe²,

Monsigny³

et al. 1986

Biochemical Journal

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Interactions of D-beta-hydroxybutyrate dehydrogenase with phospholipids were investigated by both intrinsic- and extrinsic-fluorescence approaches. The intrinsic fluorescence, mainly caused by tryptophan residues, increased upon re-activation in the presence of phospholipids bearing a positive charge, i.e. phosphatidylcholine, but decreased in the presence of non-re-activating phospholipids with a negative charge. This indicates either that the environment of tryptophan residues is affected by charges rather than by hydrophobic chains of phospholipids, or that the enzyme undergoes different conformational changes depending on the nature of the phospholipids. On the other hand, the graph of the temperature-dependence of the fluorescence intensities of the enzyme embedded in dimyristoylphosphatidylcholine liposomes exhibits a break around 21 degrees C. This indicates either that at least one tryptophan residue is closely in contact with the hydrophobic chains of phospholipids or that there is a change in the environment of tryptophan residues owing to the physical state of the phospholipids. The addition of D-beta-hydroxybutyrate apo-dehydrogenase to phospholipid liposomes containing diphenylhexatriene (a fluorescent probe) increased the diphenylhexatriene fluorescence polarization. Moreover, there was a partial fluorescence energy transfer from tryptophan to diphenylhexatriene. These results strongly favour the possibility that there is a portion of the enzyme polypeptide chain inserted into the phospholipid hydrophobic region. All these results demonstrate that D-beta-hydroxybutyrate apo-dehydrogenase interacts with both polar and hydrophobic parts of phospholipids and leads to small, but essential, conformational changes of the enzyme.

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Section: Discussionmentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

Kebbaj¹,

Latruffe²,

Monsigny³

et al. 1986

Biochemical Journal

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“…It was derived that the dissociation constant for vesicles of only phosphatidylcholine was equal to 13 x M. In agreement with these high dissociation constants titration with either vesicles did not give measurable changes in fluorescence intensity. In this regard the interaction of the exchange protein differs from that of, for example, serum apolipoproteins [ l l , 251, melittin [12] or cytochrome b5 [14] which demonstrate pronounced changes in the fluorescence spectrum upon addition of pure phosphatidylcholine vesicles. In agreement with this fluorescent behavior it has been demonstrated with ultracentrifugal flotation and gel filtration that the latter proteins [ l l -13,261 in contrast to the exchange protein [l, 61 form stable complexes with phosphatidylcholine vesicles.…”

Section: Discussionmentioning

confidence: 99%

“…HCl [13]. Similarly, denaturation of cytochrome bs by urea was retarded when lysophosphatidylcholine (molar ratio 1 : 200) was present [14]. In contrast, the interaction of the exchange protein with lysophosphatidylcholine (molar ratio 1 : 100) did not protect against denaturation by guanidine .…”

Section: Interaction With Lysophosphatidylcholine Micellesmentioning

confidence: 99%

Interaction of the Phosphatidylcholine Exchange Protein with Phospholipids

Wirtz

Moonen

1977

European Journal of Biochemistry

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The phosphatidylcholine exchange protein from bovine liver has a fluorescence emission maximum at 327 nm. Fluorescence was enhanced in the presence of vesicles containing phosphatidylcholine and various amounts of either phosphatidic acid or phosphatidylglycerol. From the increase in fluorescence it was derived that the apparent dissociation constant of the exchange protein-vesicle complex decreased with an increased vesicle content of acidic phospholipids.Fluorescence indicated that the exchange protein interacted with lysophosphatidylcholine micelles at pH 3.5, 5.9 and 8.5. The increase in fluorescence was most prominent at the acidic pH. Circular dichroism indicated that the a-helix content of the native protein was low between pH 3.6 and 8.0. Interaction with lysophosphatidylcholine micelles had a negligible effect on the secondary structure of the protein, except at pH 3.6 where distinct minima at 208 nm and 220 nm in the circular dichroic spectrum became apparent.Mammalian tissues contain a number of phospholipid exchange proteins which function as carriers of phospholipids between membranes [1 -31. The phosphatidylcholine exchange protein from bovine liver specifically carriers phosphatidylcholine [4 -61. It is presumed that upon formation of a protein-membrane complex the protein releases its bound phosphatidylcholine molecule into the interface [7]. Disruption of the complex is accompanied by incorporation of a phosphatidylcholine molecule from the membrane into the protein. Recently it has been demonstrated that the phospholipid composition of the membrane and the ionic composition of the medium govern the transfer activity of the phosphatidylcholine exchange protein [6 -81.In the present study the ineraction of the latter protein with phospholipid vesicles has been explored by fluorescence measurements. This was possible because the protein contains two tryptophan residues [9]. Fluorescence spectroscopy has been succesfully used in a number of studies on lipid-protein interactions [lo-161. In general, changes in the intrinsic fluorescence of a protein upon interaction with a lipid interface have been related to the tryptophan residues 'sensing' a different polarity of the immediate environment. From the fluorescence spectra it will be derived that, in agreement with kinetic studies [7], the dissociation constant of the exchange protein-vesicle complex decreases when the vesicles contain increasing amounts of acidic phospholipids.Previously it was found that the exchange protein can form a complex with micelles of lysophosphatidylcholine which resists disruption upon polyacrylamide gel electrophoresis [l]. The study on the interaction with these micelles has been corroborated with fluorescence and circular dichroism measurements. The latter technique in particular may give information on the effect of the interface on the secondary structure of the protein [17]. MATERIALS AND METHODSPhosphatidylcholine was isolated from egg yolk and used for the preparation of phosphatidic acid (sodium salt) and lysoph...

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“…Studies of membrane-incorporated proteins were commonly carried out on two types of model system: planar phospholipid layers and vesicular (liposome)-incorporated proteins (Ingelman-Sundberg and Glaumann, 1980;Voznesensky et al, 1990;Ramsden et al, 1996). Until recently, the kinetic parameters for lipidprotein interactions in vesicular systems were measured by fluorescence quenching of amino acid residues in the membrane fragment or by insertion of special fluorescent labels (Dufourcq et al, 1975;Leto and Holloway, 1979;Yang et al, 1981). Nowadays, the optical biosensor method is finding an increasing application in real-time studies of protein-protein (Ivanov et al, 1997(Ivanov et al, , 1999a(Ivanov et al, ,b, 2000 and planar lipid layer-protein (Ramsden et al, 1996;Salamon and Tollin, 1996a,b;Heyse et al, 1998).…”

Section: Introductionmentioning

confidence: 98%

Optical biosensor investigation of interactions of biomembrane and water‐soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers

Ivanov

Kanaeva

Gnedenko

et al. 2001

J of Molecular Recognition

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A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions.

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Study of lipid—protein interactions in membrane models: Intrinsic fluorescence of cytochrome b₅—phospholipid complexes

Cited by 43 publications

References 25 publications

Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

Interaction of the Phosphatidylcholine Exchange Protein with Phospholipids

Optical biosensor investigation of interactions of biomembrane and water‐soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers

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Study of lipid—protein interactions in membrane models: Intrinsic fluorescence of cytochrome b5—phospholipid complexes

Cited by 43 publications

References 25 publications

Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

Interaction of the Phosphatidylcholine Exchange Protein with Phospholipids

Optical biosensor investigation of interactions of biomembrane and water‐soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers

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Study of lipid—protein interactions in membrane models: Intrinsic fluorescence of cytochrome b₅—phospholipid complexes