Biochimica et Biophysica Acta 1788 (2009) 2320–2325
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m e m
Interaction of a C-terminal peptide of Bos taurus diacylglycerol acyltransferase 1 with model membranes Daniella T. Talhari a, Marli L. Moraes a,b, Priscila V. Castilho a, Osvaldo N. Oliveira Jr. a, Leila M. Beltramini a, Ana Paula U. Araújo a,⁎ a b
Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, Brazil
a r t i c l e
i n f o
Article history: Received 24 March 2009 Received in revised form 9 July 2009 Accepted 30 July 2009 Available online 6 August 2009 Keywords: Diacylglycerol acyltransferase Peptide Langmuir monolayer Liposome Oleoyl coenzyme A (OCoA) 1,2-dioleoyl-sn-glycerol (DOG) Circular dichroism Fluorescence
a b s t r a c t Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and dedicated step in the synthesis of triacylglycerol, which is believed to involve the lipids oleoyl coenzyme A (OCoA) and dioleoyl-sn-glycerol (DOG) as substrates. In this work we investigated the interaction of a specific peptide, referred to as SIT2, on the C-terminal of DGAT1 (HKWCIRHFYKP) with model membranes made with OCoA and DOG in Langmuir monolayers and liposomes. According to the circular dichroism and fluorescence data, conformational changes on SIT2 were seen only on liposomes containing OCoA and DOG. In Langmuir monolayers, SIT2 causes the isotherms of neat OCoA and DOG monolayers to be expanded, but has negligible effect on mixed monolayers of OCoA and DOG. This synergistic interaction between SIT2 and DOG + OCoA may be rationalized in terms of a molecular model in which SIT2 may serve as a linkage between the two lipids. Our results therefore provide molecular-level evidence for the interaction between this domain and the substrates OCoA and DOG for the synthesis of triacylglycerol. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Acyl-OCoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride synthesis by facilitating the linkage of sn-1,2diacylglycerol (DAG) with a long chain acyl CoA. DGAT exists in two primary isoforms, viz. DGAT1 and DGAT2 [1]; DGAT1 is most highly expressed in the small intestine and white adipose tissues, whereas DGAT2 is primarily expressed in the liver and white adipose tissues [1] where its expression is insulin-responsive. There is some evidence that the two enzymes play different roles in triglyceride metabolism. The relationship between protein structure and function in DGAT/ACAT family is still not well established owing due to the difficulty in isolating DGAT1 because of its hydrophobic character. Using a prediction domain model, Cases et al. [1] identified nine transmembrane domains and a conserved serine necessary for ACAT activity. Little is known about the DGAT1 regions involved in substrate interaction, apart from the fact that the FYXDWWN motif, highly conserved in all DGAT/ACAT family members, has been implicated in binding fatty Acyl CoA [2]. However, experimental evidence has emerged suggesting that the fatty acyl-CoA binding domain is at the N-terminus [3,4]. Also, toward the C-terminal
⁎ Corresponding author. E-mail address:
[email protected] (A.P.U. Araújo). 0005-2736/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2009.07.023
region DGAT1 possesses a putative diacylglycerol binding domain (HKWCIRHFYKP) as in protein kinase C and diacylglycerol kinases [5,6]. In order to evaluate this putative binding site in the bovine DGAT1 (NP_777118.1), we studied the interaction between the peptide corresponding to residues 379–393, denoted SIT2, with the phospholipids oleoyl coenzyme A (OCoA) and dioleoyl-sn-glycerol (DOG) since they are the natural substrates for the enzyme. Such an interaction was mimicked with membrane models comprising Langmuir monolayers [7–12] and liposomes [13–15]. The use of these membrane models to investigate the action of pharmaceutical drugs and biomolecules has been motivated by the fact that the structural framework of a cell membrane comprises a phospholipid bilayer [16] and by the difficulties in performing experiments with cell membranes in vivo. In spite of the obvious, severe simplifications, useful information can be inferred from such simple models. Indeed, one may correlate the location of a drug in a Langmuir monolayer with the pharmacological activity [17], especially in cases where penetration into the membrane is relevant for the activity. It is also possible to obtain molecular-level evidence for complexation of proteins and polysaccharides in the membrane, as is the case of chitosan that was shown to remove β-lactoglobulin from Langmuir monolayers of negatively charged phospholipids [18]. Liposomes and monolayers are complementary in terms of obviating the simplifications of membrane models. On one hand, liposomes are adequate for mimicking a bilayer and study transport
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across the membrane, which is not possible with the monolayers. On the other hand, the method with Langmuir monolayers allows control of molecular packing, which is essential for monitoring effects from guest molecules, including drugs, proteins and peptides [19–24]. Circular dichroism (CD), fluorescence emission and surface pressure–area isotherms were used to understand the SIT2 peptide– membrane model interaction and the possible biological implications are commented upon in the Final remarks. 2. Experimental procedures 2.1. Materials SIT2 (residues 379–393 from bovine DGAT1, NP_777118.1) was synthesized by Bio Synthesis, Inc.(with ≥95% purity). According to the protparam program [25], the sequence is NIPVHKWAIR HFYKP, with 15 amino acids, molecular weight of 1906.2 Da and theoretical pI of 10.29. There is no net number of negatively charged residues (Asp + Glu) and the total number of positively charged residues (Arg + Lys) is 3. To prevent any interpeptide disulfide bond formation, the residue Ala was placed instead of the original, but not conserved amongst the ACAT family [5], Cys386. The phospholipids oleoyl coenzyme A (OCoA), 1,2-dioleoyl-sn-glycerol (DOG), 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG) were purchased from Avanti Polar Lipids and were used without further purification. 2.2. Langmuir monolayers Langmuir monolayers were formed by spreading the phospholipids and peptide solutions in organic solvents (see below) onto the surface of an ultrapure water subphase, supplied by a Milli-Ro coupled to a Milli-Q system (MilliPore). The isotherm experiments were carried out in a KSV 5000 Langmuir trough in a class 10,000 clean room, with the subphase temperature kept at 22 °C using a thermostatted bath (Neslab). Equimolar solutions of phospholipids (DOG, OCoA, DPPC and DPPG) and peptide were dissolved in chloroform and methanol, respectively. An aliquot with 100 µL of DOG/SIT2, OCoA/SIT2 or DOG/OCoA/SIT2 mixtures, in different relative concentrations of the peptide, were spread onto the surface. After 15 min elapsed for evaporation of the solvent, the monolayers were compressed at a speed of 10 mm/min. Surface pressures were measured with a Wilhelmy plate provided by KSV (Finland). 2.3. Liposomes
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spectropolarimeter (JASCO Corporation, Japan) using a cylindrical 0.1 cm path quartz cuvette. The CD spectra of the solvent were subtracted to eliminate background effects and the spectra were acquired as an accumulation of 16 runs. 2.5. Steady-state fluorescence The steady-state fluorescence emission measurements were performed at 25 °C, in an ISS K2 spectrofluorimeter (ISS, Illinois, USA) using a rectangular 1 cm quartz cuvette. SIT2 samples in 10 mM phosphate buffer pH 7.5, with or without liposomes, were excited at 280 nm with a 2 mm slit, and the emission was monitored from 295 to 450 nm with a 0.5 mm slit. 3. Results and discussion In this work we evaluate a DGAT1 region (comprised by SIT2) as a promising region to be involved in the binding of diacylglycerol. This was performed by studying the interaction between SIT2 and Langmuir monolayers or liposomes made with the lipids DOG and OCoA. For comparison, experiments with SIT2 and DPPC will also be described. 3.1. Langmuir monolayers The interaction between SIT2 and DOG and OCoA was studied in Langmuir films obtained by co-spreading SIT2 with the lipids. Fig. 1 shows the surface pressure–area isotherms for DOG monolayers containing different SIT2 percentages (0.1; 0.5; 1 and 5 mol%). The pure DOG monolayer, whose molecules are not charged at a neat air/ water interface, displays an extrapolated area of ca. 120 Å2. SIT2 caused the isotherms to be considerably expanded, i.e. shifted to larger areas per molecule, with an increase in area per DOG molecule at a fixed pressure that may reach 50 Å2. The incorporation of SIT2 caused the collapse pressure to increase in comparison with the neat DOG monolayer. Here we employ the area per DOG molecule rather than the area per molecule (considering that it is a mixed monolayer), which means that all the calculations are made as if only the DOG molecules were at the interface. With such a choice, all changes caused in packing of the DOG molecules or caused by insertion of the guest molecules are readily seen. The trend of increasing expansion for increasing SIT2 concentration is broken for concentrations above 1 mol% (5 mol% of SIT2). This effect has been observed with another peptide [26], and is probably due to peptide saturation. The observed saturation at a few percent of the peptide means that large amounts of
Four compositions of liposomes were obtained with DPPC, DPPC/ OCoA (9:1), DPPC/DOG (9:1) and DPPC/DOG/OCoA (9:1/2:1/2). The phospholipids (1 mM) were dissolved in chloroform and SIT2 peptide (0.02 mM) dissolved in methanol. The chloroform and/or methanol were evaporated using liquid nitrogen until a thin film had been formed. The phospholipids and peptide were dried together. After complete evaporation of the solvents, the film was hydrated using 1 mL of preheated (40 °C, during 2 h) phosphate buffer solution (10 mM, pH 7.5). This mixture was then mixed in a vortex where multilamellar vesicles (MLVs) were formed. The liposomes were obtained by a MLVs extrusion process through polycarbonate membranes with 100 nm pores to decrease dispersity in size and formation of the unilamellar vesicles, before the spectroscopy analysis. 2.4. Far-UV circular dichroism (CD) measurements Far-UV CD measurements were performed using SIT2 peptide (0.02 mM in 10 mM phosphate buffer, pH 7.5) on its own or incorporated into liposomes solutions. Measurements were carried out in the far-UV range (195–250 nm) at 25 °C with a Jasco J-715
Fig. 1. Surface pressure–area isotherms of DOG/SIT2 monolayers containing different peptide concentrations: (solid) pure DOG, (dashed) 0.1 mol%, (dotted) 0.5 mol%, (dashed–dotted) 1 mol% and (dashed–dotted–dotted) 5 mol% of SIT2 in the monolayer.
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the peptide cannot be incorporated into the monolayer and that there may be a cooperative interaction (see comment later on), as a measurable effect occurs at very small concentrations of the peptide. In control experiments it was noted that SIT2 on its own cannot form a monolayer as most of the material spread goes into the subphase. The coupling with DOG monolayer reinforces SIT2 as a diacylglycerol binding site, for it interacts with the uncharged DOG even though it is positively charged. We have also performed several cycles of compression and decompression with the monolayers and noted that there was no significant change in the isotherms between consecutive cycles. Therefore, even if the SIT2 molecules were expelled from the interface at high pressures, they were not dissolved into the subphase in significant amounts; otherwise the subsequent surface pressure isotherm would differ from the previous one. There was another important change in the isotherms upon incorporation of SIT2, namely the appearance of further plateaus. For the pure DOG monolayer, the isotherm is relatively condensed and a long plateau appears at ca. 36–37 mN/m, which is probably due to collapse. For the isotherms containing 1 mol% or less, there was also only one plateau since there was much less peptide incorporated in the monolayer. In contrast, in the presence of a larger amount of SIT2 another phase transition occurs, as indicated in Fig. 2A. This result has been rationalized with a scheme shown in Fig. 2B, for an isotherm
Fig. 2. (A) Surface pressure isotherm depicting the possible steps in peptide conformation in a DOG monolayer, which are depicted in the scheme of figure (B).
with 5 mol% SIT2 reproduced in Fig. 2A. In Step 1, the peptide would be located in the hydrophobic region of the DOG molecules. In Step 2 the peptide is assumed to be in contact with the DOG polar head and the aqueous subphase. Therefore, the area occupied by one DOG molecule in the monolayer containing SIT2 is roughly the same as that for a pure DOG monolayer. The incorporation of the peptide is induced by the lateral pressure exerted on the monolayer, in a reversible process as subsequent surface pressure–area isotherms are identical to the first one. Note also that the distinct isotherms mean that some of the peptides were not excluded from the interface. Small quantities of SIT2 were also found to affect OCoA monolayers, as illustrated in Fig. 3. Again, the incorporation of the peptide induces isotherm expansion. For instance, the extrapolated area for a pure OCoA monolayer was ca. 40 Å2, but it increased to ca. 90–100 Å2 with 5 mol% of SIT2. Furthermore, for SIT2 concentrations above 0.5 mol%, a phase transition appeared at high pressures. Significantly, for closely packed monolayers (i.e. high pressures), all isotherms seem to coincide, which means that SIT2 molecules were expelled from the interface. Because OCoA is negatively charged and SIT2 is positively charged, one could expect a much stronger interaction than between SIT2 and DOG. However, the differences are not so large, and therefore one concludes that the interaction may also occur via other types of forces, such as hydrophobic and H-bonding. The ability of incorporation into the monolayers appears to be different for DOG and OCoA. For the OCoA monolayer, whose molecule occupies a smaller area per molecule, forming a more organized film (more closely packed), a higher amount of SIT2 could be incorporated, and saturation was not reached up to 5 mol%. A visual inspection of the isotherms for 1 and 5 mol% in Fig. 3, however, appears to indicate that saturation would eventually be reached. The DOG molecules, in contrast, occupy a much larger area at the interface, which points to a less organized film. This did not allow insertion of a high amount of SIT2, thus accounting for a saturation at a lower concentration. Because the insertion of SIT2 into the monolayers – and the film organization as well – depends on various intermolecular forces, it is not possible to determine the precise causes for the differences. A surprising result was observed for the interaction between SIT2 and mixed monolayers of DOG and OCoA, spread at the air/water interface with the same molar concentration (1:1). Fig. 4 shows that incorporation of SIT2 in 3 concentrations did not cause any significant change in the isotherms. Probably with both lipids (DOG and OCoA) in equal proportions at the interface, SIT2 cannot penetrate into the hydrophobic region, remaining in contact with the water subphase, as
Fig. 3. Surface pressure–area isotherms of OCoA/SIT2 monolayers containing various SIT2 concentrations: (solid) pure OCoA, (dashed) 0.1 mol%, (dotted) 0.5 mol%, (dashed– dotted), 1 mol% and (dashed–dotted–dotted), 5 mol% of SIT2 in the monolayer.
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Fig. 4. Surface pressure–area isotherms of DOG/OCoA/SIT2 monolayers, for 1:1 concentration of DOG and OCoA, containing different peptide concentrations: (solid) pure DOG/OCoA, (dashed) 0.5 mol% and (dotted) 1 mol% of SIT2 in the monolayer. The inset shows the suggested location for SIT2, which was based on the experimental evidence and not on molecular modeling.
indicated in the scheme shown in the inset of Fig. 4. The suggestion for the location of SIT2 was based on the identical isotherms for neat DOG + OCoA monolayers and mixed monolayers containing distinct amounts of SIT2. Such results can only be explained if SIT2 is either at the periphery of the headgroups or expelled into the subphase. Since we have evidence to suggest that SIT2 did not dissolve into the subphase in significant amounts, for consecutive compression–decompression cycles led to similar isotherms, the most probable positioning is at the periphery of the headgroups. We have also investigated the effects from adding SIT2 to mixed DOG + OCoA monolayers with various relative concentrations of DOG. Fig. 5 shows surface pressure isotherms for these mixed monolayers at a fixed concentration of 2% for SIT2. As expected, the effect from SIT2 is negligible for a 1:1 DOG:OCoA mixture, consistent with the results in Fig. 4. However, for the other concentrations, SIT2 caused the isotherms to be less expanded than for the corresponding mixed monolayer. Therefore, while SIT2 expanded both neat DOG and neat OCoA monolayers (cf. Figs. 1 and 3), it induced condensation for monolayers with small proportions of DOG in OCoA or small proportions of OCoA in DOG. The latter observation is better visualized in Fig. 6. This is probably
Fig. 5. Surface pressure–area isotherms for DOG/OCoA/SIT2 mixed monolayers at a fixed concentration of 2% for SIT2 with various relative concentrations of DOG/OCoA: (solid black) 1:0, (dashed black) 9:1, (dotted black) 3:1, (dashed dotted gray) 1:1, (dotted light gray) 1:3, (dashed light gray) 1:9 and (solid light gray) 0:1 of DOG:OCoA in the monolayer.
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because SIT2 reduces the repulsive interactions between OCoA and DOG in the mixed monolayers; such repulsion leads to the super linear behavior in the area per molecule vs. DOG concentration for small and large DOG proportions in Fig. 6. Taken all the above results together, one concludes that there is synergy in the interaction between SIT2 and DOG+ OCoA molecules, as the presence of DOG (or OCoA) prevents SIT2 molecules from being incorporated into the hydrophobic region of OCoA (or DOG) molecules. For unequal proportions of OCoA and DOG molecules, in addition to the absence of incorporation, SIT2 even caused condensation of the monolayer by reducing the repulsion between OCoA and DOG. The term “synergistic interaction” has been used in the literature to express that the effect from two or more factors is not the superimposition of the effects from the factors in separate. In our work, the synergy is clear in that the expansions caused by SIT2 on DOG and OCoA – when interacting with either of the neat monolayers – simply vanish when SIT2 interacts with the 1:1 mixed monolayer (DOG+ OCoA). This concept of synergy does not require cooperativity, even though in our measurements there is cooperativity in the interaction of SIT2 with both OCoA and DOG, as very small amounts of SIT2 were sufficient to produce a measurable effect on the isotherms. In subsidiary experiments, we also verified the interaction of SIT2 with DPPC and DPPG monolayers, two phospholipids present in abundance in cell membranes, one zwiterionic and another negatively charged. Some expansion in the DPPC isotherm was observed in Fig. 7A, which affected the phase transition from the liquid-expanded to the liquid-condensed state. This phase transition is attributed to the ordering of the hydrocarbon chains upon compression of the monolayer, and the long plateau appears because there is a significant decrease in the area occupied by each molecule as the liquid-condensed state is reached with the chains ordered. SIT2 affected the monolayer in two ways. First, a higher energy is required for inducing order in the chains, which is reflected in larger surface pressures for the phase transition. Second, this transition is not as well defined as in neat DPPC, probably owing to some disrupting of the monolayer. The changes caused by SIT2 on DPPC were nevertheless much smaller than on DOG, which is also uncharged. At high pressures, in particular, the isotherm was the same as for a DPPC monolayer. Fig. 7B indicates that SIT2 induced only small changes in the isotherms of DPPG, considerably smaller than for OCoA and DOG. At low pressures, there was a very slight increase in area per molecule, while in the liquid-condensed phase the area was even smaller than for the neat DPPG monolayer. The results with DPPC and
Fig. 6. Area per molecule for mixed monolayers with various relative concentrations of DOG:OCoA in the absence (■) and presence (●) of SIT2 2 mol% at the pressure of 20 mN/ m. The black and red lines were drawn only to guide the eyes.
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Fig. 7. Surface pressure–area isotherms of (A) DPPC/SIT2 and (B) DPPG/SIT2 monolayers containing different peptide concentrations: (solid) pure phospholipid, (dashed) 0.5 mol% and (dotted) 1 mol% of SIT2 in the monolayer.
DPPG thus corroborate the earlier inference that interactions other than the electrostatic play an important role, and points to a specific, strong interaction between SIT2 and DOG. 3.2. Liposomes The interaction of SIT2 with lipid bilayers was studied with liposomes containing different proportions of phospholipids to mimic physiological conditions. Fig. 8A shows the CD spectra of SIT2 in solution and within various types of liposomes. For SIT2 in a phosphate buffer, pH 7.5, the spectrum displayed a minimum at 197 nm compatible with a disordered structure, as frequently reported for several peptides in solution [27]. For SIT2 in the presence of DPPC and DPPC/DOG liposomes the spectra were similar to that in solution, for SIT2 probably did not bind to the liposomes or its structure was not changed upon incorporation in the bilayer. Since the liposomes are in a dominant gel phase state, SIT2 may adsorb at the surface but is not inserted in the bilayer. In the presence of DPPC/OCoA and DPPC/OCoA/DOG liposomes, however, SIT2 showed conformational change typical of proteins containing alpha and beta structures with minima at 209–216 nm (Fig. 8A). The most significant ordering was observed for SIT2 in DPPC/ DOG/OCoA liposomes. This may help explain why it may interact with lipids but not change conformation significantly. For the sake of comparison, we have also tried to produce liposomes with neat DOG or neat OCoA, but they could not be obtained, probably because they have 3 and 1 chains, respectively, which makes it difficult to obtain liposomes.
Fig. 8. Circular dichroism (A) and fluorescence emission (B) spectra of SIT2 peptide in liposomes. SIT2 only (solid) and in liposomes: DPPC/OCoA (dashed), DPPC/DOG (dotted), DPPC (dashed–dotted) and DPPC/OCoA/DOG (dashed–dotted–dotted).
The fluorescence emission spectra of SIT2 in Fig. 8B are consistent with the CD results. The spectra for SIT2 in solution and in DPPC and DPPC/DOG liposomes were similar with an emission maximum at ca. 350 nm, typical of Trp residues exposed to the solvent. The decrease in intensity is attributed to quenching caused by scattering from the liposomes. In contrast, the fluorescence spectra of SIT2 in DPPC/OCoA and DPPC/OCoA/DOG liposomes showed blue shifts of 5 nm and 22 nm, respectively (Fig. 8B). The quenching is again due to scattering by liposomes. One could in principle eliminate the effect from scattering using a control experiment with free Trp rather than the peptide (See FEBS Journal 274 (2007) 5096–5104) [28], which was not possible here because free Trp already gives a higher fluorescence intensity. Nevertheless, part of the scattering was taken into account by considering the SIT2-free liposomes. Therefore, the results with liposomes confirm the synergistic interaction between SIT2 and OCoA + DOG molecules, as in the Langmuir monolayers. The interactions will probably be electrostatic and van der Waals, though their precise nature cannot be determined with the present data. 4. Final remarks The combined use of Langmuir monolayers and liposomes as simplified membrane models has been useful to investigate the interaction between the peptide SIT2 and the lipids OCoA and DOG. From the surface pressure isotherms, SIT2 was found to expand both
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OCoA and DOG monolayers, but the same did not apply when the monolayer contained OCoA and DOG molecules in equal proportions. In the latter case, SIT2 could not penetrate into the hydrophobic region of the mixed monolayer. This was interpreted as due to a synergy in the interaction between SIT2 and OCoA and SIT2 and DOG, which also occurred in liposomes. In the CD spectra, the minima at 209–216 nm characteristic of ordered SIT2 (i.e. exhibiting alpha helices) is seen for the liposomes containing both OCoA and DOG, but not in the other cases. Similarly, in the fluorescence spectra, a large shift was only observed for SIT2 incorporated into liposomes that contained OCoA and DOG. Taken together, our results suggest that a specific C-terminal peptide (SIT2) of DGAT1 interacts with OCoA and DOG in such a way as to possibly bring together these substrates to allow for the catalytic reaction yielding triacylglycerol. Acknowledgments This work was supported by FAPESP, CNPq and Capes (Brazil). References [1] S. Cases, S.J. Smith, Y.W. Zheng, H.M. Myers, S.R. Lear, E. Sande, S. Novak, C. Collins, C.B. Welch, A.J. Lusis, S.K. Erickson, R.V. Farese Jr., Identification of a gene encoding an acyl OCoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis, Proc. Natl. Acad. Sci. 95 (1998) 13018–13023. [2] K.K. Buhman, H.C. Chen, R.V. Farese Jr., The enzymes of neutral lipid synthesis, J. Biol. Chem. 276 (2001) 40369–40372. [3] R.J. Weselake, M. Madhavji, S.J. Szarka, N.A. Patterson, W.B. Wiehler, C.L. Nykiforuk, T.L. Burton, P.S. Boora, S.C. Mosimann, N.A. Foroud, B.J. Thibault, M.M. Moloney, A. Laroche, T.L. Furukawa-Stoffer, Acyl-OCoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape, BMC Biochem. 7 (2006) 24–37. [4] R.M. Siloto, M. Madhavji, W.B. Wiehler, T.L. Burton, P.S. Boora, A. Laroche, R.J. Weselake, An N-terminal fragment of mouse DGAT1 binds different acyl-OCoAs with varying affinity, Biochem. Biophys. Res. Commun. 373 (2008) 350–354. [5] P. Oelkers, A. Behari, D. Cromley, J.T. Billheimer, S.L. Sturley, Characterization of two human genes encoding acyl coenzyme A: cholesterol acyltransferase-related enzymes, J. Biol. Chem. 273 (1998) 26765–26771. [6] C.L. Yen, S.J. Stone, S. Koliwad, C. Harris, R.V. Farese Jr., Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis, J. Lipid Res. 49 (2008) 2283–2301. [7] L.P. Cavalcanti, O. Konovalov, I.L. Torriani, Lipid model membranes for drug interaction study, Eur. Biophys. J. Biophys. 35 (2006) 431–438. [8] G. Borissevitch, M. Tabak, O.N. Oliveira Jr., Interaction of dipyridamole with lipids in mixed Langmuir monolayers, Biochim. Biophys. Acta 1278 (1996) 12–18. [9] T.F. Schmidt, L. Caseli, T. Viitala, O.N. Oliveira Jr., Enhanced activity of horseradish peroxidase in Langmuir–Blodgett films of phospholipids, Biochim. Biophys. Acta: Biomembranes 1778 (2008) 2291–2297.
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