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-Monoolein In Vitro1
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742
2To whom correspondence should be addressed at. E-mail: Eb112{at}umail.umd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß-isomers of MO. The turbidity of increasing concentrations
of aqueous dispersions of -MO and ß-MO in the presence and absence
of BSA was measured in triplicate by absorption spectrophotometry.
Aqueous dispersions of [13C1]MO and
[13C1]MO/BSA mixtures at molar ratios of 1:1,
3:1 and 5:1 were analyzed in duplicate by [13C]nuclear
magnetic resonance (NMR) at pH 7.4 and 36°C. BSA bound significantly
more ß-MO than -MO at 15 min: 5.4 ± 0.42 and 3.3 ± 0.60 mol MO/mol BSA, respectively (P < 0.05).
[13C]NMR spectra of the 1:1 molar ratio of
[13C1]MO/BSA exhibited a single carbonyl peak
at 175.19 ppm, whereas spectra of 3:1 and 5:1 molar ratios exhibited
three peaks between 172 and 174 (ppm), each distinct from carbonyl
resonances of either [13C1]MO dispersed in
water, 176.72 (ppm) or BSA alone. The intensities of individual peaks,
but not their chemical shift values, varied between 3:1 and 5:1 molar
ratios, indicating that BSA has at least three MO binding sites and may
bind up to five molecules of MO per molecule. This study confirms that
serum albumin binds MO in vitro and supports the theory that albumin
transports monoglycerides produced by lipoprotein lipase hydrolysis of
triglyceride.
KEY WORDS: • monoolein • serum albumin • [13C]NMR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), 2) MG remain on chylomicron remnants and VLDL
or transfer from TG-rich lipoproteins to HDL and are taken up by
the liver (El-Maghrabi et al. 1978a and 1978b
) or
3) MG are bound and transported by serum albumin in a manner
similar to FA (Arvidsson and Belfrage 1969
,
Thumser et al. 1998
). Although albumin-FA binding
has been extensively investigated, very little information has been
presented on albumin-MG binding (Arvidsson and Belfrage 1969
, Thumser et al. 1998
). On the basis of
solution partitioning, Arvidsson and Belfrage (1969)
reported that human serum albumin binds approximately seven molecules
of monoolein (MO). However, by measuring changes in the fluorescence of
tryptophan, Thumser et al. (1998
) determined that human
serum albumin binds approximately two or three molecules of MO and that
MO is bound at sites distinct from oleic acid. In addition, from
competitive binding and displacement studies with oleic acid, MO and
other fluorescent ligands, the authors concluded that albumin bound MO
in subdomain IIIA (Thumser 1998
). However, because
neither of the previous studies specified the MO isomer composition
used, it is likely to have been predominantly the -isomer. No data
have been reported for the interaction of serum albumin and the more
physiologically relevant ß-MG isomer. In addition, both of these
studies relied on relatively indirect methods to measure albumin-MO
binding.
[13C]Nuclear magnetic resonance (NMR) is a
noninvasive method that has been used to directly investigate the
number, location and strength of albumin binding sites for FA
(Cistola et al. 1987a and 1987b
, Hamilton et al. 1991
, Parks et al. 1983
). FA carbonyls have
unique chemical shift values when they are bound to albumin,
solubilized in PC vesicles or dispersed in water due to the different
physical and chemical environments of the FA carbonyl in each
physiological compartment (Hamilton 1989
,
Hamilton and Cistola 1986
). Also, FA present at
different binding sites of albumin exhibit different chemical shift
values (Cistola et al. 1987a and 1987b
, Hamilton et al. 1991
, Parks et al. 1983
). The objective
of this study was to directly investigate the interaction of bovine
serum albumin (BSA) and MO using [13C]enriched
MO and to examine potential differences in the interaction of BSA with
the - and ß-isomers of MO.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The glycerolysis procedure described by Choudhury (1960
)
was modified as follows: 150 mg of
1,1,1-[13C3]triolein
(99%; Cambridge Isotope Laboratories, Andover, MA), 50 µL of 1 mol
NaOH/L and 65 µL of glycerol were combined in a glass vial, flushed
with N2 and heated to 160°C for 4–6 h under a
vacuum with constant stirring. Aliquots of 30 mg of glycerolysis
products were dispersed in 500 µL of benzene, applied to a presoaked
5-g silica cartridge and separated by column chromatography using an
adaptation of the methods described by Horwitz (1975
).
Acylglycerols were eluted in order of increasing polarity by the
addition of 80 mL each of benzene, benzene/ethyl ether [90:10 (v/v)]
and ethyl ether. Eluate solvents were evaporated under
N2, and lipid fractions were weighed, flushed
with N2 and stored at -80°C. Acylglycerols in
each fraction were identified by thin-layer chromatography (TLC) by
comparison with authentic lipid standards (Nuchek Prep, Elysian, MN)
using a mobile phase of benzene/ethyl ether/ethyl acetate/acetic acid
[80:10:10:0.2 (v/v/v/v)] (Christie 1973
). Then 10-µL
aliquots of [13C1]MO
stock solution were applied to TLC plates impregnated with 1.2% boric
acid and separated into the - and ß-isomers using a mobile phase
of chloroform/methanol [98:2 (v/v)].
Measurement of MO.
After TLC, MO and FA were extracted from silica, transmethylated in 3
mL of acetyl chloride methanol [1:15 (v/v)] containing 50 µg of
heptadecanoic acid (C:17:0) as an internal standard and held at 60°C
for 2 h. Fatty acid methyl esters were extracted into 500 µL of
hexane containing 10 µg of methyl laurate (C:12:0) and quantified by
gas chromatography (GC) on a Hewlett-Packard 5890 Series II gas
chromatograph equipped with a DB-23 capillary column (J&W Scientific,
Folsom, CA). The concentration of MO was calculated from the micrograms
of methyl oleate corrected for percentage methylation and extraction.
The [13C1]MO was 
85% -MO and 
15%
ß-MO and the acyl purity was 99% as determined by TLC, GC and
13C NMR. The concentration of
[13C1]MO stock solutions was determined by GC
before all sample preparation.
Preparation of MO and phosphatidylcholine (PC) dispersions.
Two mL of 0.05 mol Tris/L heated to 50°C was added to 2–4 mg of
liquid -MO or ß-MO (1-monooleoyl-rac-glycerol and
2-monooleoyl-glycerol; Sigma Chemical, St. Louis, MO) or egg yolk
PC and immediately mixed with a vortex mixer. The mixtures were
sonicated for 10 min in pulse mode with 15-s cycles (550 Sonic
Dismembrator; Misonix, Farmingdale, NY). A nondispersible mesophase of
MO (Larsson 1989
) formed and stuck to the sides of the
vial in 
65% of the MO dispersions. The dispersed MO was aspirated
and transferred to a clean glass vial, and the MO concentration was
determined by GC. MO dispersions ranged from 1.51 to 3.55 mmol/L.
Preparation of [13C1]MO dispersions.
[13C1]MO was transferred to a glass vial, dried under N2 and dispersed in 1 mL of 10 mmol potassium phosphate and 100 mmol EDTA per L to yield a final concentration of 6.0 mmol/L, pH 7.4. The mixture was sonicated for 60 min in pulse mode with 6-s cycles. Next, it was centrifuged for 10 min at 500 x g to remove titanium particles that may have chipped off of the sonicator probe tip.
Preparation of serum albumin.
Essentially FA-free (0.005%) BSA of 96% purity (Sigma
Chemical) was dissolved in 0.05 mol Tris/L, pH 7.4, to yield
concentrations of 0.075 mmol/L for turbidity measurements. BSA used for
[13C]NMR measurements was dissolved in 60% (v/v)
D2O (Aldrich Chemical Company, Milwaukee, WI) to yield a
160 g/L stock solution. After the determination of protein
concentration according to a modified Lowry assay (Peterson 1977
), potassium phosphate and EDTA were added to the stock
solution to final concentrations of 10 and 100 mmol/L, respectively.
BSA solutions were stored at 5°C and used within 30–45 d. One-mL
aliquots of BSA were acidulated to <pH 4, extracted with
chloroform/methanol [2:1 (v/v)] and analyzed by TLC and GC for FA as
described here and found to be >99% FA free.
Measurement of turbidity.
The turbidity measurements used to demonstrate a binding interaction
between BSA and lipids were adapted from the procedure described by
Wang et al. (1993
). Appropriate amounts of -MO,
ß-MO or PC dispersions were combined with 1 mL of 0.075 mmol BSA/L
and Tris buffer to yield samples of 2 mL total volume and with molar
ratios of MO/BSA or PC/BSA ranging from 1:1 to 11:1. Samples were mixed
with a vortex mixer and equilibrated for 15 and 60 min at room
temperature, and the absorbance at 450 nm was measured using a
Spectronic 21D (Busch & Lomb, Rochester, NY) spectrophotometer. Each
sample was measured twice, and the mean of these measurements was
calculated. Experiments were replicated three times for each isomer.
Control experiments with PC were replicated twice. The concentration of
MO and PC in samples ranged from 0.0375 to 0.413 mmol/L. The absorbance
data were plotted against the molar ratio of lipid/BSA as well as the
absolute concentration of lipid in each sample to demonstrate
differences between the number of BSA binding sites for - and ß-MO
isomers.
[13C1]MO/BSA.
Appropriate volumes of dispersed [13C1]MO stock solution were transferred to 5 mm NMR tubes. The solvent was evaporated under N2, and 0.8–0.9 mL BSA stock solution was added to yield a final concentration of 1.2 mmol BSA/L and 1.2, 3.6 or 6.0 mmol [13C1]MO/L, producing [13C1]MO/BSA molar ratios of 1:1, 3:1 and 5:1, respectively. Each sample was prepared in duplicate, and [13C]NMR spectra were acquired for each replicate.
[13C]NMR spectroscopy.
[13C]NMR spectra were acquired on a Bruker AM400
spectrometer (Billerica, MA) at 36°C, using a 60° pulse (6.57
µs), 16,384 data points, receiver gain of 1600 and either
heteronuclear broadband or inverse-gated decoupling at 1 W.
Acquisitions per spectrum are shown in the figure legends. Acquisition
times for each experiment ranged from 12 to 16 h. Spectra were
processed with baseline correction and exponential line broadening of 3
Hz before Fourier transformation unless otherwise noted. The chemical
shift values (
) were measured digitally with a precision of ±0.1
ppm and referenced to 3-(trimethylsilyl)-1-propanesulfonic acid, sodium
salt at 0.0 ppm when present or the 
-lysine/ß-leucine peak of BSA
at 39.54 ppm when present (Parks et al. 1983
). For
comparison between spectra, all chemical shift values were then
normalized to the -lysine/ß-leucine peak of BSA at 39.54 ppm. The
line width at half-height (
1/2) of selected
resonances was measured manually after processing the spectra without
line broadening. The intensity values of selected peaks were the
average of two observations in cm, normalized to 100-cm height of the
highest peak in the protein aromatic region at 130 ppm. The standard
deviations of the intensity values were <10%.
Statistical analysis.
Turbidity measurements were analyzed using join-point regression
analysis (Draper and Smith 1983
) for two linear line
segments with different slopes (SAS Institute, Cary, NC). The
join-point (point where the slope changes) with standard error and
the upper and lower confidence intervals (L1 and L2, respectively) at
95% probability are reported. Difference in join point between isomers
was analyzed using t tests (P < 0.05) at each time point.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The turbidity data are presented as absorption versus the molar ratio
of lipid/BSA as well as the absolute concentration of lipid. This
comparison was used to demonstrate differences between - and
ß-isomers of MO. The absorbance of both - and ß-MO increased
linearly with concentration and decreased significantly in the presence
of BSA (Fig. 1
). The point at which the slope of the line changed is the "join
point." The join point for -MO occurred at a concentration
equivalent to 3.3 ± 0.60 mol of -MO/mol BSA at 15 min; upper
(L1) and lower (L2) confidence intervals were 2.15 and 4.53,
respectively; and increased slightly to 3.5 ± 0.64 mol -MO/mol
BSA (L1 = 2.17, L2 = 4.74) at 60-min equilibration (data not
shown). At 15 min, the join point for ß-MO was 5.4 ± 0.42 mol
ß-MO/mol BSA (L1 = 4.58, L2 = 6.26) and decreased slightly
to 5.2 ± 0.44 mol ß-MO/mol BSA (L1 = 4.34, L2 = 6.10)
at 60-min equilibration (data not shown). The join points for - and
ß-MO at 15 min were significantly different at 15-min (P
< 0.05) but not 60-min equilibration (P = 0.065).
In contrast, the absorbance of PC dispersions increased linearly with
concentration and did not change in the presence of BSA at either 15 or
60 min (Fig. 2
).
|
|
The distribution of isomers in the
[13C1]MO produced was
85% -MO and 15% ß-MO, which is typical of MG produced via
glycerolysis (Choudhury 1960
). The carbonyl region of
the [13C]NMR spectra of the
[13C1]MO dispersed in water (170–185 ppm)
contained a single symmetric peak at 176.72 ppm with a line width of
30.1 Hz (Fig. 3E
). The same region of the [13C]NMR spectrum of
BSA contained a broad hump centered at 175.0 ppm (Fig. 3D)
, which is
the overlap of carbonyl carbon signals from glutamine, asparagine and
the peptide backbone of BSA, and a small sharp peak at 181.04 ppm,
which is the glutamic acid carboxyl (Parks et al. 1983
).
The glutamic acid carboxyl resonance is approximately the same height
as the overlapping carbonyl and carboxyl carbons.
|
When [13C1]MO was mixed
with BSA, four different peaks with chemical shift values distinct from
those of [13C1]MO in
water were detected (Table 1
and Fig. 3A
3B
3C
.). At a 1:1 molar ratio of
[13C1]MO/BSA, a single sharp
[13C1]MO carbonyl peak was observed at 175.19
ppm (Fig. 3C)
in each replicate. At both 3:1 and 5:1 molar ratios of
[13C1]MO/BSA, three
different peaks for
[13C1]MO, referred to as
peaks a, b and c, were present (Figs. 3A
and 3B)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MO, based on turbidity measurements at 15 min. However,
the changes in sample turbidity between 15 and 60 min indicate that the
samples may not have been equilibrated at the earlier time point.
Alternatively, the changes in sample turbidity over time could have
resulted from altered binding due to the spontaneous acyl migration
between ß- and -MO isomers. Studies in our laboratory determined
that 15–20% of the total ß-MO present in an aqueous dispersion will
undergo acyl migration within 1 h at room temperature
(Lyubachevskaya and Boyle-Roden 2001
). Although we
did not measure the rate of - to ß-MO acyl migration, the
equilibrium ratio of -MO/ß-MO is 88:12 (Choudhury 1960
); thus, pure -MO would also equilibrate to a mixture of
- and ß-MO. Due to the indirect nature of light scattering, the
exact number of binding sites for each isomer can be estimated but not
conclusively determined. Interpretation of the join point from
turbidity plots as the stoichiometric binding ratio of albumin to a
ligand were based on studies by Wang et al. (1993
). In
our studies, turbidity resulted from light scattered by aggregates or
particles of MO. A decrease in sample turbidity could be caused by
either a decrease in the number of particles scattering light or by a
change in the size of particles within a sample (Everett 1989
). The differences in absorption of -MO, ß-MO and PC
at equivalent concentrations in the absence of BSA were probably due to
differences in the size of particles within each dispersion. There was
no change in turbidity of MO dispersions up to 24 h after
preparation (data not shown). The changes in turbidity occurred only on
addition of BSA. Thus, the decreased turbidity of MO and BSA mixtures
was attributed to BSA binding MO, thereby reducing the total number of
MO aggregates or particles present in a sample. PC vesicles were used
as a negative control because they scatter light but do not bind to BSA
(Peters 1996
). As expected, the addition of BSA to
increasing concentrations of PC had no effect on turbidity.
The difference in chemical shift values as well as line widths for
carbonyls in the
[13C1]NMR spectra
indicate that [13C1]MO
mixed with BSA was in a different magnetic microenvironment than when
dispersed in water. At the 1:1 molar ratio of
[13C1]MO/BSA, MO
exhibited a more downfield chemical shift value, indicating that the MO
carbonyl group either was located in a more hydrophilic environment or
experienced a different degree of hydrogen bonding with the protein
compared with MO at 3:1 and 5:1 molar ratios. It is probable that the
first several methylene carbons of the MO acyl chain were not involved
in hydrophobic interactions in this binding site, allowing for greater
hydration of the carbonyl. MO bound to BSA at higher molar ratios was
more "protected" from water as indicated by the up-field
chemical shift. The disappearance of the downfield resonance (175.19
ppm) at higher molar ratios may have resulted from a conformational
change of BSA in the presence of 1 mol of MO. Conformational changes
of serum albumin on the addition of FA are well documented
(Curry et al. 1998
, Peters 1996
) and have
been predicted after the addition of MO to BSA (Thumser 1998
). Because of this conformational change, the binding site
present at the 1:1 molar ratio was either no longer available at the
higher molar ratios or had a significantly altered magnetic environment
and physical conformation.
The presence of only three distinct resonances at both 3:1 and 5:1
molar ratios of
[13C1]MO/BSA indicates
that BSA has a minimum of three binding sites for MO. Alternatively,
more than three binding sites for MO may exist but have chemical shift
values that overlap, making them indistinguishable by the methods used
in this study. The latter explanation is supported by both the solvent
partitioning data of Arvidsson and Belfrage (1969)
and
the turbidity data presented here indicating that BSA binds five
molecules of ß-MO. In addition, the presence of three distinct peaks
indicates that the rate of MO exchange between these three binding
sites on BSA was slow on an NMR time scale. The absence of a carbonyl
peak with a chemical shift value equivalent to
[13C1]MO dispersed in
water in the 3:1 and 5:1 molar ratio samples indicates that all of the
[13C1]MO present was
bound to BSA.
The carbonyl line widths of
[13C1]MO in
[13C1]MO/BSA mixtures
were quite similar to published values for
[13C1]FA in
[13C1]FA/BSA mixtures
(6–15 Hz), indicating that MO experiences a similar degree of
molecular motion as FA when bound to BSA (Cistola et al. 1987s
, Parks et al. 1983
). FA are bound to BSA
primarily through hydrophobic interactions between the FA acyl chain
and amino acid residues lining the side of binding sites, whereas ionic
interactions between carbonyls and amino acid residues at the face of
binding sites also contribute to the stability of FA/BSA complexes
(Bojesen and Bojesen 1996
, Peters 1996
,
Spector 1975
). The less stringent hydrophobic
interactions along the acyl chain contribute to the relatively high
mobility of FA molecules bound to BSA, as indicated by 13C
NMR line width data (Cistola et al. 1987a
, Parks et al. 1983
). Arvidsson and Belfrage (1969
)
proposed that MO associates with serum albumin via primarily
hydrophobic interactions. The data presented here support their
conclusion that MO associates with BSA in a manner very similar to its
association with FA. The free hydroxyl groups of MO are proposed to
interact with charged amino acid residues at the face of binding sites
via H-bonding, contributing to the stability of MO/BSA complexes.
The observation that BSA initially binds more ß-MO than -MO
indicates that H-bonding or polar interactions contribute to MO/BSA
binding. The difference in the number of binding sites estimated for
-MO and ß-MO may be related to how the free hydroxyl groups of the
glycerol are oriented for H-bonding with amino acid residues at the
face of binding sites. Just as the ionization state of the FA carboxyl
affects FA/albumin binding (Cistola et al. 1987b
), the
positioning of free hydroxyl groups on the MG glycerol may affect
MG/albumin binding.
In a fasting state, the concentration of MG in circulation is several
hundred times less than that of FA (Fielding 1993
).
However, immediately after the hydrolysis of TG in the circulation, the
concentrations of both MG and FA increase rapidly, with the
concentration of MG approaching one half that of FA at the point of
lipolysis. Studies have shown that MG could be absorbed into cells at
and perhaps adjacent to the point of lipolysis (Scow et al. 1976
) or be carried to the liver via chylomicron remnants or
other lipoproteins (El-Maghrabi et al. 1978a and 1978b
).
The ability of serum albumin to bind multiple molecules of MG indicates
that MG could also be delivered to tissues at some distance from the
point of TG lipolysis. In addition, because albumin is a transporter of
numerous pharmaceutical agents, the competition of both FA and MG for
binding sites could alter the concentration of active or unbound drug
in the circulation. In summary, this study confirmed that serum albumin
binds both - and ß-isomers of MO, has a minimum of three separate
binding sites for MO and can bind at least five molecules of MO per
protein molecule. Thus, serum albumin may transport MG produced in vivo
by the hydrolysis of TG-rich lipoproteins.
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ACKNOWLEDGMENTS |
|---|
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FOOTNOTES |
|---|
3 Abbreviations used: BSA, bovine serum albumin; [13C1]MO, [13C]carbonyl-enriched (99%) mono-olein; FA, fatty acid; MG, monoglyceride; MO, mono-olein; NMR, nuclear magnetic resonance; PC, phosphatidylcholine.
Manuscript received July 20, 2000. Initial review completed August 23, 2000. Revision accepted November 30, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Arvidsson E. O., Belfrage P. Monoglyceride-protein interaction: The binding of monoolein to native human serum albumin. Acta. Chem. Scand. 1969;23:232-234[Medline]
2. Bojesen E., Bojesen I. N. Albumin binding of long-chain fatty acids: Thermodynamics and kinetics. J. Phys. Chem. 1996;100:17981-17985
3. Choudhury R. B. R. The preparation and purification of monoglycerides. I. Glycerolysis of oils. J. Am. Oil Chem. Soc. 1960;37:483-486
4. Christie W. W. Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids 1973:152-154 Pergamon Oxford
5.
Cistola D. P., Small D. M., Hamilton J. A. Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. I. The filling of individual fatty acid binding sites. J. Biol. Chem. 1987a;262:10971-10979
6.
Cistola D. P., Small D. M., Hamilton J. A. Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. II. Electrostatic interactions in individual fatty acid binding sites. J. Biol. Chem. 1987b;262:10980-10985
7. Curry S., Mandelkow H., Brick P., Franks N. Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Nat. Struct. Biol. 1998;5:827-830[Medline]
8. Draper N., Smith H. Applied Regression Analysis 2nd ed. 1983:255-256 Wiley New York.
9. El-Maghrabi M. R., Waite M., Rudel L. L., Sisson P. Hydrolysis of monoacylglycrol in lipoprotein remnants catalyzed by the liver plasma membrane monoacylglycerol acyltransferase. J. Biol. Chem. 1978a;53:974-998
10. El-Maghrabi M. R., Waite M., Rudel L. L. Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase. Biochem. Biophys. Res. Commun. 1978b;81:82-88[Medline]
11. Everett D. H. Basic Principles of Colloid Science 1989:102-103 Royal Society of Chemistry England.
12. Fielding B. A., Humphreys S. M., Frayn K. N. Mono- and diacylglycerol concentrations in human plasma in relation to lipoprotein lipase activity. Biochem. Soc. Trans. 1993;21:235S
13.
Hamilton J. A. Medium-chain fatty acid binding to albumin and transfer to phospholipid bilayers. Proc. Natl. Acad. Sci. USA 1989;86:2663-2667
14.
Hamilton J. A., Cistola D. P. Transfer of oleic acid between albumin and phospholipid vesicles. Proc. Natl. Acad. Sci. USA 1986;83:82-86
15.
Hamilton J. A., Era S., Bhamidipati S. P., Reed R. G. Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin. Proc. Natl. Acad. Sci. USA 1991;88:2051-2054
16. Horwitz W. In: Official Methods of Analysis of the Association of Official Analytical Chemist 12th ed. 1975:515-516 AOAC Washington, D.C.
17. Larsson K. Cubic lipid-water phases: Structures and biomembrane aspects. J. Phys. Chem. 1989;93:7304-7314
18. Lyubachevskaya G., and Boyle-Roden E. Kinetics of 2-monoacylglycerol acyl migration in model chylomicra. Lipids 2000;35:1353-1358[Medline]
19.
Parks J. S., Cistola D. P., Small D. M., Hamilton J. A. Interactions of the carboxyl group of oleic acid with bovine serum albumin: A 13C NMR study. J. Biol. Chem. 1983;258:9262-9269
20. Peters T. All About Albumin: Biochemistry, Genetics and Medical Applications 1996 Academic Press San Diego.
21. Peterson G. L. Simplification of the protein assay method of Lowry et al. Which is more generally applicable?. Anal. Biochem. 1977;83:346-348[Medline]
22.
Scow R. O., Blanchette-Mackie E. J., Smith L. C. Role of capillary endothelium in the clearance of chylomicrons. Circ. Res. 1976;39:149-162
23. Spector A. S. Fatty acids binding to plasma albumin. J. Lipid Res. 1975;16:165-179[Abstract]
24.
Thumser A. E., Buckland A. G., Wilton D. C. Monoacylglycerol binding to human serum albumin: Evidence that monooleoylglycerol binds at the dansylsarcosine site. J. Lipid Res. 1998;39:1033-1038
25. Wang CS, Bass H, Whitmer R., McDonathy W. J. Effects of albumin and apolipoprotein C-II on the acyl-chain specificity of lipoprotein lipase catalysis. J. Lipid Res. 1993;34:2091-2098[Abstract]
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) or absence
(•) of 0.0375 mmol BSA/L. MO dispersions were diluted to
0.0375–0.413 mmol/L with Tris buffer, pH 7.4, and mixed with BSA to
yield MO/BSA molar ratios from 1:1 to 11:1. Data points with error bars
are the means of three replicates ± SEM; data points
without error bars are the means of duplicates.
