(Received for publication, November 7, 1994; and in revised form, December 9, 1994)
From the
High density lipoprotein (HDL) phospholipid (PL) fatty acyl
chain composition has been proposed to affect the ability of HDL to
participate in the first step of reverse cholesterol transport. To
examine the effects of PL fatty acid chain length and degree of
unsaturation in this process, reconstituted HDL (rHDL) particles were
made with human apolipoprotein (apo) A-I and PL containing fatty acid
chains from 14 to 18 carbons in length, which were either fully
saturated or unsaturated in one or both chains. These particles were
characterized structurally and for their ability to promote free
(unesterified) cholesterol (FC) efflux from cells growing in culture.
The discoidal rHDL particles were homogeneous and exhibited similar
hydrodynamic diameters (10.4 ± 1.0 nm) indicating that apoA-I
forms similarly sized discs with a variety of PL. Measurements of
particle surface charge, apoA-I -helix content, and conformational
stability indicated that the conformation of apoA-I varies among the
particles. These conformational effects on apoA-I are consistent with
the PL fluidity influencing the interaction between the amphipathic
-helical segments and PL acyl chains. Differential scanning
calorimetry demonstrated that the physical state of the rHDL PL at 37
°C varied according to acyl chain length and degree of
unsaturation; the FC efflux efficiencies for particles with PL in
either the gel or liquid crystal states were determined. The ability of
the rHDL to accept cellular FC depended on the physical state of the PL
in the rHDL. Liquid crystal PL formed the most efficient FC acceptor
particles exhibiting a maximal efflux velocity (V
) of 12-14% release of total cellular FC
per h. Gel-phase PL formed inefficient rHDL acceptors with a V
of about 3%/h. A similar hierarchy of FC
efflux efficiency was noted when either mouse L-cells or rat Fu5AH
hepatoma cells were used as the FC donors. Furthermore, this hierarchy
was found to be due to the characteristics of the PL and not due to
variable apoA-I conformation because protein-free, small unilamellar
vesicles made with the same PL exhibited similar relative efflux
capabilities. Generally, the ability of a given rHDL particle to accept
cellular FC was related to rHDL PL acyl chain length and degree of
unsaturation; decreases in PL acyl chain length and increases in chain
unsaturation tended to result in more efficient FC acceptor particles.
These results suggest that rHDL acceptor particles that contain highly
fluid surfaces sequester FC molecules that have diffused from the cell
plasma membrane at a significantly faster rate than those containing
highly organized lipid surfaces with restricted PL acyl chain mobility.
This information forms a basis for understanding the role of lipid
content in the structural and functional diversity of HDL.
High density lipoprotein (HDL) ()exhibits substantial
structural heterogeneity (1) and recent evidence indicates that
this lipoprotein class may be functionally heterogeneous as well.
Distinct HDL subspecies may be involved in different aspects of reverse
cholesterol transport, the process by which excess peripheral cell
cholesterol (FC) is returned to the liver for catabolism(2) .
Castro and Fielding (3) have demonstrated that a minor HDL
charge subspecies, pre-
HDL, may be an important mediator of the
first step of reverse cholesterol transport, the transfer of FC from
cells to HDL. It has been proposed (3) that this particle
initially accepts peripheral cell FC and subsequently transports it to
larger HDL species that contain lecithin-cholesterol acyltransferase
where it can be esterified. Rader et al.(4) have
shown that other HDL particles exhibit an increased rate of clearance
from the plasma by the liver. Still other HDL particles appear to be
able to interact with plasma enzymes such as lecithin-cholesterol
acyltransferase(5) . Although the various HDL subspecies are
known to have variable lipid and protein compositions, little is known
about the structural features of the HDL subspecies that give rise to
functional diversity.
Previously, we have investigated the role of
HDL apolipoprotein structure in the transfer of FC from cells to
HDL(6) . The current study focuses on the PL component of HDL.
Several studies have shown that the ability of a given particle to
accept cellular FC is related to the amount of PL present in the
particle(6, 7) . In addition, there is evidence that
the types of phospholipids present in HDL may be important. Sola et
al.(8) demonstrated that diet-induced changes in the
fatty acyl chain composition of HDL lead to differences in
the ability of HDL
to remove FC from cultured fibroblasts.
Increases in the percentage of saturated acyl chains in the HDL
reduced its ability to accept cellular FC relative to HDL
that was enriched in unsaturated acyl chains. This effect was
attributed to decreases in the lipid fluidity of the HDL
surface as a result of the increased percentage of saturated acyl
chains(9) . Dietary modifications such as these have been
suggested to have an effect on the FC transfer to HDL in
vivo(10) , although such studies cannot distinguish
between the lipid modification of HDL and modifications of peripheral
cell membranes which can also affect FC transfer to HDL(11) .
Our objective in this study was to test the hypothesis that the PL acyl chain content can affect the ability of HDL particles to remove FC from cells. A second objective was to examine the effect of PL acyl chain content on the structure of associated apoA-I. In order to avoid complicating factors such as variable PL:protein ratios, the presence of lipids other than PL, variable protein contents, and large changes in particle size that can occur in studies using in vivo modified HDL, we reconstituted well-defined rHDL particles that were similar in size as well as composition. A variety of synthetic PL containing saturated acyl chains from 14 to 18 carbons in length as well as length-matched PL containing unsaturated acyl chains were complexed to human apoA-I using a sodium cholate removal technique. The resulting particles were characterized with respect to composition, size, charge, lipid physical state, and apoA-I conformational stability. The relative efficiencies of the rHDL in removing FC from cultured cells were determined over a range of concentrations. The results show that the physical state of the PL molecules in the rHDL particles significantly affects the ability of the particles to remove cell FC. The information derived from this study has implications for understanding the role of HDL lipids in the first step of reverse cholesterol transport.
Table 1shows the compositions and diameters of particles from four independent sets of reconstitutions. On a molar basis, the final PL:protein molar ratios were in the range from 75 to 100:1 with each particle containing two molecules of apoA-I per particle. The exceptions were the particles containing DSPC and PSPC which ranged from 55 to 60:1 (PL:apoA-I) and contained 3 molecules of apoA-I. The particle diameters fell generally within a 1.5-nm range with an overall average of 10.4 nm by PAGGE and 10.1 nm by electron microscopy. POPC/apoA-I and OPPC/apoA-I particles exhibited identical sizes and compositions suggesting that the position of unsaturated acyl chains did not affect the binding of apoA-I to PL. DSPC/apoA-I and PSPC/apoA-I particles had the largest hydrodynamic diameters despite their reduced PL:apoA-I ratio; the increased disc diameters were most likely due to the presence of the third molecule of apoA-I.
The D term must be interpreted with caution when
comparing apoA-I conformational stabilities in different rHDL
particles. Previous reports have suggested that PL complexed to apoA-I
may inhibit the binding of GdnHCl molecules to the target
protein(15, 31) . Therefore, apparent increases in D when studying rHDL containing various PL may be artifactual
because of the interference of GdnHCl binding by the PL. The analysis
used here accounts for the variable activities of GdnHCl to give a
standard free energy of denaturation
(
G
) to describe the stability of the
-helical segments of apoA-I. The
n term in Table 3is the number of GdnHCl molecules that bind to each
molecule of apoA-I during the denaturation. The denaturation of free
apoA-I exhibited a high
n, whereas apoA-I complexed to LC
PL exhibited a 2-fold lower value; an even lower value was evident when
apoA-I was complexed to gel-phase PL (DPPC and PSPC). These results are
consistent with the concept that PL interferes with GdnHCl binding to
apoA-I and that gel-phase PL acyl chains perturb this interaction more
than fluid PL. When these factors are taken into account, apoA-I in
rHDL containing LC PL exhibited similar
G
values to free apoA-I, whereas the apoA-I in the gel-phase rHDL
particles was less stable.
Figure 1:
Time course of
[H]free cholesterol efflux from mouse L-cell
fibroblasts to discoidal rHDL containing various PL acyl chains. Mouse
L-cell fibroblasts that had been trace labeled with
[
H]free cholesterol in 22-mm tissue culture wells
were incubated for 6 h at 37 °C in a humidified incubator (5%
CO
) with 1.0 ml of test medium containing 0.5% bovine serum
albumin, 1.0 µg/ml Sandoz compound 58035, and the rHDL containing
the indicated PL at 100 µg of PL/ml. The rHDL particles were:
POPC/apoA-I (
), DOPC/apoA-I (
), DMPC/apoA-I (
),
DSPC/apoA-I (
), PSPC/apoA-I (
), DPPC/apoA-I (
), and
SM/apoA-I (
). The vertical axis indicates the fraction of initial
labeled free cholesterol that remained in the cell at the designated
times. Each point represents the mean of six cell wells. The error
bars represent 1 S.D. All curves were obtained by fitting the
entire time course to the model for tracer equilibration between two
pools (see ``Methods'').
Fig. 1shows clear differences between the various rHDL with
the POPC/apoA-I and DOPC/apoA-I particles being the most effective
acceptors and DSPC/apoA-I and PSPC/apoA-I particles being the least
effective. The half-times (t, corrected for FC
efflux to control medium) for FC efflux from L-cells to the rHDL made
with DOPC, POPC, PSPC, DMPC, DPPC, DSPC, and SM were 12.8 ± 1.4,
10.6 ± 0.9, 32.4 ± 3.8, 25.0 ± 3.6, 22.2 ±
1.0, 32.2 ± 6.0, and 13.8 ± 3.0 h, respectively.
OPPC/apoA-I and POPC/apoA-I particles showed identical kinetics
indicating that the position of unsaturated acyl chains was not
important in determining FC efflux efficiency (data not shown). The PL
that were in the LC state at 37 °C promoted faster FC efflux than
those in the gel-phase. Furthermore, the relative efflux efficiency was
inversely related to the T
(Table 1) of the
PL composing the particle. For example, the DMPC/apoA-I disc, although
in a LC state at 37 °C, did not promote as much FC transfer as
DOPC/apoA-I and POPC/apoA-I discs. A similar relationship was noted
among the gel-phase rHDL. To determine the efflux over a range of
concentrations, FC efflux rate constants were derived for each particle
at each of five concentrations ranging from 20 to 500 µg of PL/ml
of medium (Fig. 2). Kinetic analysis of the time courses at the
highest acceptor particle concentrations indicated that the predicted
equilibrium distribution of cellular FC was generally similar for both
LC- and gel-phase rHDL (about 70% of cell FC was present in the
acceptor pool at equilibrium). The relative FC efflux rate differences
observed at 100 µg of PL/ml (Fig. 1) persisted at 5-fold
higher acceptor particle concentrations. To estimate the maximal efflux
velocity (V
) that was attained by these
complexes, the initial rates of cellular FC efflux (occurring during
the first 2 h) were plotted as a function of particle concentration as
proposed by Hofstee(32) . This treatment linearized the data so
that a V
could be estimated by extrapolating the
data to the condition of infinitely high acceptor concentration (for a
detailed discussion of the application of this analysis to FC efflux,
see (6) and the Appendix). The average (n = 6) V
values (percent of cellular FC released per h)
for the rHDL made with DOPC, POPC, PSPC, DMPC, DPPC, DSPC, and SM were
14.0 ± 1.7, 12.0 ± 2.2, 2.5 ± 0.2, 6.2 ±
1.1, 4.9 ± 0.7, 3.2 ± 0.6, and 8.8 ± 2.4,
respectively. The FC efflux rates were slightly increased in separate
incubations in which the cell wells were stirred at 500 rpm on an
orbital shaker (2 mm radius), but the differences in FC efficiency that
are shown in Fig. 2were found to persist whether the cell
plates were stirred or left quiescent (data not shown). These data show
that the V
values for the various rHDL did not
converge to a common value.
Figure 2:
The concentration dependence of the FC
efflux rate constants (k) from mouse L-cell
fibroblasts to discoidal rHDL containing various PL acyl chains. The
incubation conditions symbols were the same as described in the legend
to Fig. 1. The vertical axis shows the average FC efflux rate
constant (k
) derived by fitting an
equation that describes tracer equilibration between two pools (see
``Methods'') to an experimental time course. Thus each data
point is derived from an entire 6-h time course. Each point represents
the mean of six cell wells from two separate experiments. The error
bars represent 1 S.D. The values shown are corrected for the FC
efflux to control medium.
To determine if the relative FC efflux
efficiencies are a function of the cell type used, similar FC efflux
experiments were performed using rat Fu5AH hepatoma cells which are
known to release FC at a faster rate than L-cells in response to
PL-containing particles (33) (Fig. 3). In this case, the
corrected t values for the efflux of Fu5AH FC to
the rHDL made with DOPC, POPC, PSPC, DMPC, DPPC, DSPC, and SM were 7.3
± 0.3, 5.5 ± 0.3, 33 ± 6.0, 11.2 ± 1.0,
12.2 ± 0.7, 63.4 ± 9.3, and 8.6 ± 0.3 h,
respectively. Relative to L-cells, the Fu5AH cells released FC more
rapidly to the LC rHDL at a concentration of 100 µg of PL/ml ( Fig. 1and Fig. 3). However, the FC transfer to gel-phase
(DSPC/apoA-I and PSPC/apoA-I) particles was the same or slower than
from L-cells. The same ranking of the abilities of the various rHDL
particles to remove FC was observed with the two cell types. This
indicates that the relative abilities of these complexes to remove
cellular FC was a particle-dependent phenomenon and was not related to
the particular cell type used.
Figure 3:
Time courses of
[H]free cholesterol efflux from rat Fu5AH
hepatoma cells to discoidal rHDL containing various PL acyl chains. The
incubation conditions and symbols were the same as described in the
legend to Fig. 1. The vertical axis indicates the fraction of
initial labeled free cholesterol that remained in the cell at the
designated times. Each point represents the mean of three cell wells.
The error bars represent 1 S.D. All curves were obtained as
described in Fig. 2.
It was possible that the nature of
the PL in the rHDL determined the particle's ability to accept
cellular FC although it was also possible that the conformation of the
apoA-I on the surface of the particle could be a determinant. To
distinguish between these possibilities, two of the PL types were
selected to make protein-free, small unilamellar vesicles (SUV). POPC
and DMPC were used because they exhibited large differences in efflux
efficiency when complexed to apoA-I (Fig. 2) and preliminary
work has established that both PL form well-defined SUV that do not
degrade or fuse under the conditions of the FC efflux assay (data not
shown). Fig. 4compares the concentration dependence of FC
efflux from L-cells to apoA-I-containing rHDL and to protein-free SUV
made with POPC and DMPC. At 500 µg of PL/ml, the POPC/apoA-I disc
had a k of about 12% cell FC/h and the DMPC/AI had
a k
of about 6% cell FC/h, a 2-fold difference. At
the same concentration, the POPC SUV exhibited a lower rate of 2.5%/h
and the DMPC SUV had a k
of about 1.1%/h, about a
2-fold difference. Since the 2-fold difference in efflux efficiency
between the apoA-I-containing discs is also observed between the SUV
made with the same PL, it follows that the relative order of efflux
efficiency seen in Fig. 2was due to the nature of the PL
present in the rHDL and not to the conformation of the apoA-I on the
surface of the particle.
Figure 4:
Comparison of the concentration dependence
of the FC efflux rate constants (k) from
mouse L-cell fibroblasts to apoA-I-containing discoidal rHDL and SUV
made with either POPC or DMPC. Panel A shows the data for rHDL
discs (replotted from Fig. 2) and panel B shows the
data for SUV. Incubation conditions were the same as those for Fig. 1. Each point represents three cell wells and the error
bars represent 1 S.D. The values shown are corrected for the FC
efflux to control medium.
The rHDL FC efflux efficiency can be
related to the length and degree of saturation of the acyl chains of
the rHDL PL. Increases in the rHDL PL acyl chain length and degree of
saturation apparently decrease the FC efflux efficiency of rHDL. In
terms of acyl chain length of rHDL PL, the FC efflux hierarchy is DMPC
(2 14:0) > DPPC (2
16:0) > DSPC (2
18:0) (Fig. 1Fig. 2Fig. 3). In the case of degree of
unsaturation, the FC efflux ranking is DOPC (2
18:1) > DSPC
(2
18:0), and PSPC (16:0 18:0) <POPC (16:0 18:1).
The results of this study show that both the structure and function of discoidal rHDL are significantly affected by PL acyl chain length and degree of unsaturation because these parameters directly determine the physical state of the lipid that composes the particle. The physical state and hence fluidity of the PL at 37 °C affects the conformation of associated apoA-I molecules and the ability of an rHDL particle to remove FC from cells. These two effects are discussed in turn below.
Although the various PL affect the surface charge of the rHDL particles, this parameter does not appear to correlate with the physical state of the associated PL. Since the various PC molecules in the rHDL particles have the same net charge at a given pH, any changes in particle valence must arise from conformational changes in the resident apoA-I molecules that lead to alterations in the ionization states of charged amino acid residues(27) . Although the exact molecular nature of these changes cannot be determined, the variations in the rHDL particle charges reported in Table 2are consistent with the types of conformational effects discussed in the preceding paragraph.
Fig. 2shows that, at low rHDL concentrations (<100 µg of PL/ml), rHDL containing LC PL appear to be more efficient than those containing gel-phase PL. Since the diffusion coefficients of the various rHDL particles in the extracellular medium are expected to be similar because of their similar sizes (Table 1) and the diffusion coefficient of FC is the same in all cases, the aqueous diffusion model argues that the number of FC-rHDL collisions should be similar for the various rHDL. Thus, the observed differences must be due to a difference in the fraction of FC-rHDL collisions that result in incorporation of the FC molecules into the rHDL PL surface (effective collisions). It is likely that the percentage of effective FC collisions is higher for rHDL containing the LC PL than those containing the gel-phase PL. This seems reasonable because the incoming FC molecule must overcome the stronger cohesive interactions between the gel-phase PL acyl chains in order to incorporate into the acceptor PL surface.
At high acceptor
concentrations (>500 µg of PL/ml), the FC efflux differences
between the rHDL particles persist and the V values indicate substantially different maximal FC efflux
velocities among the rHDL of different PL content. This result is
surprising because, according to the aqueous diffusion mechanism,
different acceptor particles should exhibit similar V
values that are related to the tendency of FC to desorb from the
PM (k
). Since the relative FC efflux differences
are similar at high and low acceptor concentrations, it is reasonable
to infer that the rationale described for the case of low acceptor
concentrations can explain the differences between rHDL containing the
various PL at all rHDL concentrations. If this is correct, it follows
that the reported V
k
[DC], because no terms are
included that describe the interaction of FC molecules with the
acceptor rHDL particles. This suggests that the V
values that are measured in this study are apparent values that
may not represent the true maximal rate of FC efflux at high
concentrations of acceptor. The simplest explanation for this
discrepancy is that the concentration of the rHDL particles at the
surface of the cells (where most of the FC transfer is expected to
occur) is limited to a concentration that is: (a) distinct
from the bulk medium concentration and (b) below that required
to saturate the cellular FC efflux. This difference between the bulk
medium and cell surface concentrations is expected to be the same for
all of the rHDL particles used in this study because of their
comparable size. Under these conditions, the rate constant (k
) for FC diffusion to and incorporation into the
acceptor particle is expected to affect the FC efflux rate as it does
in the case of low acceptor concentrations. Consequently, the
differences in FC efflux noted at high concentrations can be explained
by the different fraction of effective collisions between desorbed FC
and the various rHDL (i.e. the effective FC collision rate is
lower with gel-phase rHDL). It is interesting that the equilibrium
distribution of FC among the cell and acceptor pools is generally
similar for rHDL made with both LC and gel-phase lipids, suggesting
that those particles near the cell surface can exchange with those in
the bulk medium so that the entire acceptor pool is available to accept
cell FC regardless of any limitations in the concentration close to the
cell surface. Apparently, the nature of the rHDL PL only affects the
rate at which a given rHDL can accept FC and not its capacity to
solubilize FC.
It is likely that the complexity of the PM of living
cells accounts for the lower concentration of rHDL particles at the
cell surface. The most obvious complications inherent in cellular FC
transfer systems include the unstirred water layer at the cell
surface(41) , the cell glycocalyx, and the convoluted nature of
the PM(42) . The thickness of the unstirred water layer over
most planar cells is between 300 and 800 µm(41) .
Westergaard and Dietschy (43) have demonstrated that this value
can be decreased to a minimum of 100 µm with vigorous stirring.
Although the effect of stirring on the thickness of the unstirred water
layer was not determined in this study, the increased V values observed with stirring of the cell
plates support the assertion that the values that are obtained with
quiescent cell plates are apparent values. At this point, the cell
characteristic(s) that shelters the region proximal to the PM from
further increases in acceptor particle concentration remain to be
elucidated. Experiments using acceptor particles of varying size are
needed to further investigate this phenomenon. In addition, the nature
of the donor PM can be changed. For example, the cells can be grown in
suspension culture or PM vesicles can be used as FC donors to address
the effects of cell surface organization on FC efflux.
Alternatively, if the physical basis for the differences between the
gel- and LC-phase rHDL FC efflux efficiencies is not due to differences
in the effective collision rates (i.e.V = k
[DC]), then one
is forced to conclude that the different V
values in this study arise from differential effects of the
various rHDL on either k
or
[DC] (or both). A change in the FC desorption rate (k
) could be caused by differential interactions
of the various rHDL with the cell PM. Such an interaction could be
mediated by the apolipoprotein component of the rHDL (6) or by
exchange of the PL component of the rHDL into the cell PM. The
observation that SUV exhibit similar differences in FC efflux
efficiency as apoA-I-containing rHDL (Fig. 4) indicates that the
observed differences are due to a property of the acceptor PL. While
rHDL PL undoubtedly exchanges into the cells to some extent over the
course of the efflux incubation(44, 45) , the rate of
transfer of PL is generally slow compared to the transfer of
FC(2) ; cells that have been incubated with DOPC (46) and SM (47) liposomes for much longer than the 6-h
incubations employed here show no significant change in the fatty acid
content of the PM. Thus, during the time frame of our studies, it is
unlikely that cell-accumulated PL could sufficiently modify the PM to
an extent that has been shown to have noticeable effects on the rate of
FC efflux(11) . It could be argued that the various rHDL have
differential access to regions of the PM, altering the size of the PM
FC pool ([DC]) that is participating in efflux.
However, the similar diffusional properties of the various rHDL of
comparable size suggests that all of the rHDL particles have equal
access to the various regions of the cell PM. Again, more information
from experiments where acceptor particle size is systematically changed
is required to distinguish between these possibilities.
A full
understanding of the different V values reported
for the various rHDL awaits further study. Nevertheless, it appears
that rHDL which contain gel-phase PL allow a lower fraction of
colliding FC molecules to incorporate into the PL surface than those
containing LC-phase PL. It is interesting that the SM/apoA-I discoidal
rHDL exhibits a faster rate than predicted from its physical state.
Since the SM used in this study was isolated from bovine brain, this
may be due to the heterogeneous nature of the various acyl chains
causing defects that may facilitate the incorporation of incoming FC
molecules into the bilayer. It is unlikely that HDL particles
exhibiting the large differences in PL fluidity observed with the
particles reconstituted with purified PL (Table 1) occur in
vivo. However, several lipids that are common constituents of HDL
are known to significantly affect the fluidity of lipid surfaces (e.g. FC and SM)(2) . In addition, the PL fatty acyl
composition of lipoproteins and cell membranes reflect the dietary
intake of saturated fat (8, 44) and such changes are
known to have subtle but measurable effects on the fluidity of plasma
lipoproteins(9) . These changes may affect the ability of HDL
particles to accommodate FC molecules that have desorbed from
peripheral cells. The current study using simplified systems forms a
basis for understanding the impact of HDL lipid composition on the
process of reverse cholesterol transport and offers insight into the
mechanism of FC efflux from cells.
The transfer of lipid molecules between donor and acceptor particles by diffusion has been studied extensively (for reviews, see (38) and (49) ). It is generally agreed that, depending upon the concentrations and nature of the donor and acceptor particles, and the nature of the transferring molecule, complex kinetics with different reaction orders can be observed. For instance, it has been shown that spontaneous transfer of long-chain phospholipids can occur by both a first-order, monomer desorption process and a parallel, concentration-dependent, transfer process in which the rate of lipid monomer desorption from the donor particle is enhanced via transient donor-acceptor collisions(39, 50) ; the contribution of the donor-acceptor collision-mediated process to phospholipid transfer is enhanced at higher concentrations of particles. Interestingly, the transient donor-acceptor collisional contribution is not observed for FC transfer in vesicle systems under conditions where the phospholipid transfer rate is significantly increased by formation of transient vesicle-vesicle complexes(50) . In light of this, the following kinetic model for FC transfer does not include the latter effect; also, such a process is not consistent with the zero-order kinetics seen for FC transfer from cells at high acceptor concentrations. The hyperbolic dependence of FC transfer rate on acceptor concentration is explained in terms of a kinetic scheme in which a first-order reaction to form a FC intermediate state is followed by a second-order interaction with the acceptor particle to transfer the FC molecule from the intermediate state to the acceptor particle.
The scheme for donor particles D and acceptor particles (A) can be written in terms of the two steps (see (39) and (50) -52).
where DC and AC represent FC in the donor and
acceptor particles, respectively. C is FC in the
intermediate state and the k values are the rate constants for
the steps indicated. Under initial velocity conditions where k
0, the rate of formation of C
from mass action kinetics is given by .
At the steady state equilibrium, d[C]/dt = 0 so
that
From , the initial velocity (v) of the transfer reaction
where the terms in square brackets are concentrations at time t 0.
describes the hyperbolic dependence
of v on the acceptor particle concentration. When k[D]
k
[A], there is first-order
dependence of v on acceptor concentration because v = k
[A] at
constant donor concentration; the apparent rate constant k
= k
k
[DC]/k
[D]
contains collisional rate constants k
and k
. When k
[A]
k
[D], or simply
[A]
[D] when both donor and
acceptor species are similar so that similar on-rates for FC are
expected (i.e.k
k
), v =
k
[DC] which indicates that v is
independent of the acceptor concentration [A] and
zero-order kinetics occur at constant donor concentration. The rate
constant k
describes the diffusion of FC from the
donor particle into the intermediate state C
; any
factors that modulate the packing of FC molecules in the surface of the
donor particle can influence k
(cf. (38) ). Mixed kinetics occur when k
[D]
k
[A].
The molecular events
involved in FC transfer cannot be determined unambiguously from the
kinetics but some mention of the physical process is worthwhile. FC
transfer has been explained in terms of two limiting situations: either
an aqueous diffusion mechanism where the desorption rate k is limiting or a collision-mediated mechanism
where collisions between donor and acceptor particles are involved. In
fact, both models can fit the same kinetic scheme of a first-order
formation of an intermediate followed by a second-order transfer of FC
from the intermediate state to the acceptor particle so that there is a
hyperbolic dependence on [A] (cf. Fig. 2). The nature of C
has been the
subject of much debate, it has been considered variously as FC monomers
in the aqueous phase (for a review, see (38) ) or FC molecules
partially dissociated from the donor particle(52) . When
[A] is low, the rate of FC transfer is proportional
to the number of collisions between FC molecules in the intermediate
state and acceptor particles so that v is proportional to
[A]. The collision event to form the acceptor-FC
complex AC could occur in either of the above loci, with this being
influenced by the nature of the donor and acceptor particles and the
interaction between them. It should be noted that direct contact
between the donor and acceptor particles is not obligatory because FC
can transfer between donors and acceptors separated by a semipermeable
membrane (for a review, see (38) ). At high
[A], v is zero-order with respect to
[A] and the transfer rate is limited by k
at a constant donor concentration. This rate is
affected by the structure of the donor particle and also by
donor-acceptor interactions such as might occur in
apolipoprotein-containing systems(53) .