(Received for publication, April 8, 1994; and in revised form, December 15, 1994)
From the
Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 ± 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an intergral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.
We reported that the absorption of cholesterol by brush border
membrane vesicles (BBMV) ()made from rabbit small intestines
is protein-mediated(1, 2) . It was shown to be a
second-order reaction regardless of the nature of the donor particle (1, 2, 3) . The reaction mechanism involves
collision-induced transfer of cholesterol between donor and acceptor.
We also reported cholesterol absorption to be most efficient from mixed
bile salt micelles the composition of which resembles the in vivo situation(2) . From these micelles cholesterol absorption
was about 10
-10
times faster than from small
unilamellar phospholipid vesicles or mixed micelles consisting of
lysophosphatidylcholine, egg phosphatidylcholine, and cholesterol
(60:38:2, weight ratio)(2, 3) .
After subjecting BBMV to proteolytic treatment, cholesterol absorption was shown to be a passive process characterized by half-times of the order of hours even with bile salt micelles as donor particles(2) . It involves cholesterol desorption from the donor, diffusion of monomeric cholesterol through the aqueous phase, and incorporation of cholesterol into the bilayer of BBM(1) . The passive process is a true first-order reaction indicating that cholesterol desorption is the rate-limiting step(1) .
Proteolysis of BBM proteins can be mediated by adding extrinsic proteinases, but also by activating intrinsic proteinases(2) . Proteolysis by both extrinsic and intrinsic proteinases results in a significant reduction or loss of protein-mediated cholesterol absorption by BBM. At the same time it liberates water-soluble lipid exchange proteins(1, 2, 3, 4) . These proteins catalyze cholesterol (1) and phosphatidylcholine (PtdCho) exchange between two populations of small unilamellar phospholipid vesicles(2, 3, 4) . Furthermore, some of these proteins bind both cholesterol and PtdCho as shown by ESR spin labeling (1, 3) . Assuming that the water-soluble lipid exchange proteins resulting from the proteolytic treatment are part of the original integral membrane protein(s) responsible for cholesterol and PtdCho absorption, the purification and characterization of these proteins is desirable.
Here we describe the release of water-soluble lipid exchange proteins from BBMV by the action of intrinsic proteinases and the purification of one of these lipid exchange proteins. The purified protein was used to produce polyclonal antibodies in guinea pig which were used to study the immunological relationship between various lipid exchange proteins and to shed light on the question whether these proteins are related to the cholesterol receptor present in the BBM.
1,2-Dipalmitoyl-sn-phosphatidyl[N-methyl-H(U)]choline
and the spin-labeled PtdCho,
1-palmitoyl-2-(5-doxylpalmitoyl)-sn-phosphatidylcholine
(5-doxyl-PtdCho), were synthesized as described in (3) and (5) , respectively. All lipids used in this work were pure by
TLC standard, the two spin labels were also pure by carbon, hydrogen,
and nitrogen microanalysis.
Figure 1:
A, gel filtration pattern on
Sephadex G-75 SF of supernatant proteins liberated from rabbit small
intestinal BBMV as described previously(2, 3) . About
10 ml of a protein solution (11 mg/ml) in PBS were applied to the
Sephadex G-75 SF column (36 4.6 cm; void volume V
= 170 ml, total volume V
= 600 ml) and proteins were
eluted with the same buffer at a flow rate of 32 ml
h
. Fractions of
6 ml were collected and
analyzed for lipid exchange activity: PtdCho (-), cholesterol
(+++), and phosphatidylinositol exchange
(
). B, gel filtration pattern on Sephadex
G-75 SF of supernatant proteins liberated by intrinsic proteinases from
human small intestinal BBMV. About 1 ml of a protein solution (20
mg/ml) in PBS was applied to a Sephadex G-75 SF column (53.5
1.0 cm; V
= 16 ml, V
= 44 ml) and proteins were eluted with the same
buffer at a flow rate of 3.3 ml h
. Fractions of
0.44 ml were collected and analyzed for protein(- - -) and PtdCho
exchange (-). C, gel filtration on Bio-Gel P-10 of peak
3 proteins. Supernatant proteins released from rabbit small intestinal
BBMV were first fractionated on Sephadex G-75 SF and peak 3 proteins
were pooled and concentrated. About 50 µl of protein solution (6
mg/ml) in PBS were applied to the Bio-Gel P-10 column (18.5
0.9
cm; V
= 4.8 ml, V
= 11.7 ml) and proteins were eluted with the same
buffer at a flow rate of 2 ml h
. Fractions of about
0.15 ml were collected and analyzed for protein(- - -) and PtdCho
exchange (-). In all three panels lipid exchange is expressed as
% of the total amount of lipid present in the donor prior to the start
of the exchange reaction.
Polyclonal antibodies against peak 1 lipid
exchange protein were raised in guinea pig. Good use was made of the
observation (see below) that this protein binds spin-labeled cholestane (Fig. 6G). Upon incubation of peak 1 proteins with
mixed taurocholate micelles (sodium taurocholate/oleic
acid/monooleoylglycerol/3-doxyl-5-cholestane, 88:6:3.5:2.5, weight
%; total lipid/spin label mole ratio, 30:1) in PBS (0.14 M NaCl, 0.01 M sodium phosphate, pH 7.3, containing 2.5
mM EDTA and 0.02% NaN
), at room temperature
overnight, tiny needles crystallized out consisting of spin label and
protein. The crystalline pellet was washed twice by centrifugation at
14,000
g for 10 min and resuspension in Hepes/Tris
buffer, pH 7.3, followed by repeated washing cycles using 2%
deoxycholate and finally 10% SDS. Proteins extracted with these
detergents were separated by SDS-10% PAGE, protein bands at about
100-120, 90, and 60 kDa were cut out and the proteins were
electroeluted from the gel following the Bio-Rad instruction manual.
The proteins thus obtained were injected into guinea pigs to produce
polyclonal antibodies. Only polyclonal antibodies raised against the
100-120-kDa protein were active and inhibited cholesterol and
PtdCho exchange between two populations of SUV induced in the presence
of peak 1 proteins.
Figure 6:
ESR
spectra of 5-doxyl-PtdCho and 3-doxyl-5-cholestane present in
different environments were recorded at room temperature on a Varian
X-band spectrometer (model E-104A). A, 5-doxyl-PtdCho
suspended in PBS at
1 mg of lipid/ml. B, a film of
5-doxyl-PtdCho deposited on the glass wall of a round-bottom flask was
exposed to a solution of peak 1 proteins (38 mg/ml) in PBS at 4 °C
overnight. Multilamellar liposomes were spun down by centrifugation at
12,000
g for 10 min and the ESR spectrum of the
supernatant was recorded. C, egg PtdCho (4 mg/ml = 5.33
mM) containing 3-doxyl-5
-cholestane at a lipid/spin label
mole ratio of 127 was dispersed in PBS. D, egg lyso-PtdCho (20
mg/ml = 0.040 M) containing 5-doxyl-PtdCho at a mole
ratio of
200:1 dispersed in PBS. E, sodium deoxycholate
micelles containing spin-labeled PtdCho (mole ratio
200) dispersed
in PBS at 20 mg/ml (=0.048 M). F, sodium
deoxycholate micelles spin-labeled with 3-doxyl-5
-cholestane (mole
ratio
150) and dispersed in PBS at 20 mg/ml (=0.048 M). G, sodium deoxycholate micelles spin-labeled with
3-doxyl-5
-cholestane (mole ratio = 85) and dispersed in PBS
were mixed with peak 1 protein(s) to final concentrations of bile salt
(4.6 mg/ml = 0.011 M) and peak 1 protein of 30 mg/ml,
and the ESR spectrum was immediately
recorded.
Proteins present in peak 3 of Fig. 1A were pooled, concentrated, and applied to a Bio-Gel P-10 column in order to test the resolution of these proteins obtained on Sephadex G-75. The elution pattern of peak 3 proteins on Bio-Gel P-10 consisted of a main, fairly symmetric protein peak and two minor ones at larger elution volumes (Fig. 1C). The PtdCho exchange activity was associated with the main protein peak, the shift observed in the peak position reflects the inhomogeneity of the proteins present in the main peak (cf. PAGE pattern in Fig. 2). The peak position of the PtdCho exchange activity was very close to that of ribonuclease A corresponding to an apparent molecular mass of 13.7 kDa.
Figure 2:
Cation exchange chromatography (FPLC) of
peak 3 proteins on Mono S HR 5/5. Proteins of peak 3 (Fig. 1A) were pooled, exhaustively dialyzed against
0.01 Hepes/Tris buffer, pH 7.3, containing 0.3 MD-mannitol, 5 mM EDTA, and 0.02% NaN and concentrated to
2 mg of protein/ml. The resulting
protein solution was centrifuged at 100,000
g for 20
min, filtered through a sterile Millipore filter, and 2.3 mg of protein
were applied to the Mono S column. The column (5
0.5 cm) was
run with buffer A (5 mM sodium phosphate, pH 7.0, 0.3 MD-mannitol) and after the pass-through peak, proteins
bound to the column were eluted with a Na
gradient at
0.5 ml/min using buffer A plus 1 M NaCl. All buffers were
degassed and filtered through sterile Millipore filters prior to use.
Protein (
); PtdCho exchange (
); NaCl concentration (- - -).
Panel on the right: SDS-15% PAGE patterns of the proteins present in
peak 3 prior to cation exchange chromatography (lane A) and
protein eluted from the Mono S column at V
= 27 ml (lane B). SDS-PAGE was carried out
using the Mini-Protean II dual slab cell from Bio-Rad. Proteins were
visualized by silver staining. Bio-Rad low range protein standards were
used and the position of apparent molecular masses in kDa of these
standards are marked on the right.
Figure 3:
Immunoblotting of BBMV, supernatant
proteins, and proteins present in peaks 1, 2, and 3 using IgG of guinea
pig antisera raised against the 13-kDa protein. Supernatant proteins
from rabbit small intestinal BBMV were prepared as described in Refs. 2
and 3. BBMV (30 µg of protein), total supernatant proteins (30
µg), proteins of peak 1 (30 µg), peak 2 (30 µg), and peak 3
(7 µg) were electrophoresed through SDS-15% PAGE (A) and
SDS-10% PAGE (B) and transferred in a Bio-Rad blotting apparatus to
PVDF (Immobilon-P from Millipore) using 0.025 M Tris buffer,
pH 8.3, containing 0.19 M glycine, 0.01% SDS, and 15%
CHOH as the blotting buffer and 30 V overnight at room
temperature or 300 V for 2 h at 4 °C. Immunostaining of the
transferred protein bands was carried out following the protocol of van
Heusden et al.(11) . Su, total supernatant
proteins; pk1, pk2, and pk3 refer to peak 1, 2, and 3
proteins, respectively. The numbers on either side of the
immunoblots represent the apparent molecular masses in kDa of the
marker proteins used.
Figure 4:
Effect of polyclonal antibodies on PtdCho
and cholesterol exchange between two populations of SUV mediated by
different soluble lipid exchange proteins that were liberated from
rabbit small intestinal BBMV(2, 3) . Donor vesicles
consisting of egg PtdCho, egg PtdOH, (85:15, mole ratio), and a trace
amount of [H]dipalmitoyl-PtdCho or
[
H]cholesterol and acceptor vesicles of pure egg
PtdCho, both dispersed in Hepes/Tris buffer, pH 7.3, were mixed to give
final lipid concentrations of 0.1 and 1 mg/ml, respectively. PtdCho
exchange was mediated by peak 1 proteins (27 µg of protein/ml) in A, by peak 2 proteins (37 µg of protein/ml) in B,
and peak 3 proteins (32 µg of protein/ml) in C. Closed
symbols refer to PtdCho exchange in the presence of IgG of
antisera raised against the purified 13-kDa protein. Open symbols in panels A and C refer to cholesterol exchange
in the presence of IgG of antisera raised against the 120-kDa protein
of peak 1. Different closed symbols refer to different experimental
series.
Figure 5:
A,
time course of PtdCho absorption by rabbit small intestinal BBMV. SUV
of egg PtdCho labeled with [H]dipalmitoyl-PtdCho
as donor vesicles and BBMV as the acceptor, both dispersed in
Hepes/Tris buffer, pH 7.3, were mixed to final concentrations of 0.71
mg of lipid/ml and 8.4 mg of protein/ml (4 mg of total lipid/ml),
respectively. The time courses of the PtdCho absorption by BBMV in the
absence and presence of IgG (
4 mg of protein/ml) of guinea pig
anti-13 kDa antisera (closed symbols) are compared. The
equilibrium concentration x
of the donor was
calculated from the lipid pools of donor (b) and acceptor (a): x
= 100
b/(a + b) = 22%. The lipid
concentration [a] of the acceptor was experimentally
determined to be [a] = [total
lipid]
0.6, where [total lipid] is the total
lipid concentration of BBMV(21) . The lipid concentration
[b] of the donor is equal to the analytical
concentration of egg PtdCho. B, the kinetic data of panel
A were linearized according to ln[(x- x
)/(x
-
x
)] = -k
[(a + b)/a]t, where x
= 100% at t = 0
and x and x
are the radioactivities
(in %) in the donor at time t and at equilibrium (t
), respectively. Closed symbols, in the
absence of antibody; open circles, in the presence of IgG at
4 mg of protein/ml. Different closed symbols represent
different experiments. The solid lines are least-squares fits
to the experimental data points (r
=
0.997).
Where x, x, and x
are the fractions or percentages of
exchangeable lipid in the donor at time 0, t, and at
equilibrium, respectively, a and b are the effective
lipid pools of acceptor and donor, respectively.
The effect of the
IgG fraction of the guinea pig antisera raised against the 13-kDa
protein on PtdCho uptake by rabbit small intestinal BBMV is included in Fig. 5. IgG at 4 mg of protein/ml produced a 30% inhibition of
the PtdCho uptake by BBMV (Fig. 5A) while preimmune
serum had a slightly stimulating effect on PtdCho uptake (data not
shown). The inhibitory effect was linear with IgG concentrations (data
not shown) and more importantly, it exhibited a time lag. The onset of
inhibition occurred reproducibly after a 30-min reaction of BBMV
with IgG (cf. open circles,Fig. 5A).
Linearization of the kinetic data obtained in the presence of IgG
yielded two straight lines (open symbols of Fig. 5B). Up to
30 min the experimental points lay
on the straight line fitted to the solid symbols representing
PtdCho uptake in the absence of IgG. The slope of the straight line
fitted to the data points after
30 min (Fig. 5B)
yielded a pseudo first-order rate constant k
= 0.27 h
indicating that the rate of
PtdCho uptake was reduced by a factor of about 4. Preincubation of BBMV
with IgG for 2.5 h produced slightly but significantly smaller values
than k
= 0.27 h
. Due to
the relatively large scatter of the initial data points it is uncertain
whether or not preincubation eliminated the time lag.
When
peak 1 proteins (23 mg/ml) were incubated with sodium deoxycholate
micelles containing 5-doxyl-PtdCho, the ESR spectrum changed
instantaneously from one characteristic of the spin label in the bile
salt micelle with 2 A = 36 G (Fig. 6E) to the immobilized spectrum of Fig. 6B with 2 A
= 60 G. Similarly,
when peak 1 proteins (
30 mg/ml) were incubated with sodium
deoxycholate micelles containing 3-doxyl-5
-cholestane, there was
also an instantaneous change from the three-line spectrum
characteristic of the cholestane spin label in deoxycholate micelles
with 2 A
= 38 ± 1 G (Fig. 6F) to a near rigid-limit powder spectrum with 2
A
= 66 ± 1 G (Fig. 6G, Table 1). This 2 A
value indicated very tight
binding of the cholestane spin label to peak 1 proteins. The kinetics
of the binding of the two spin labels from deoxycholate micelles was
characterized by half-times of the order of seconds. Inspection of the
spectra in Fig. 6, F and G, shows that the
spectrum in Fig. 6G is a composite one: in addition to
the near rigid-limit powder spectrum there is a second minor component
of a three-line spectrum arising from some cholestane spin label
molecules still being present in bile salt micelles. By progressively
increasing the concentration of peak 1 proteins the intensity of the
near rigid-limit powder spectrum (Fig. 6G) increased at
the expense of the three-line spectrum (Fig. 6F)
indicating that the equilibrium is pushed toward the lipid-protein
complex (data not shown).
The 13-kDa protein behaved qualitatively
similar to peak 1 proteins. Incubation of sodium cholate micelles
containing 3-doxyl-5-cholestane with the 13-kDa protein (1.7
mg/ml) in PBS yielded a composite ESR spectrum similar to that shown in Fig. 6G with a maximum hyperfine splitting of 2
A
= 63 ± 2 G (Table 1). However,
in this case the intensity of the three-line spectral component arising
from the spin label in deoxycholate micelles clearly exceeded that of
the near rigid-limit powder spectrum (data not shown). The behavior of
peak 1 and the 13-kDa protein is contrasted by that of peak 2 proteins.
When peak 2 proteins (up to
20 mg/ml) were incubated with sodium
cholate micelles containing 3-doxyl-5
-cholestane, no binding of
spin-labeled cholestane to peak 2 protein(s) was detected.
Figure 7:
Electron micrographs of immunogold-stained
BBMV. In a, BBMV from rabbit small intestines were subjected
to immunogold staining using the IgG fraction of the guinea pig
antisera against the rabbit 13-kDa protein. In b the BBMV were
treated with guinea pig preimmune serum as a control. BBMV suspended in
Hepes/Tris buffer, pH 7.3, were treated as described in (8) and (9) . The IgG fraction of antisera against the
13-kDa protein was diluted to 1 mg of protein/ml with blocking
buffer (PBS containing 50 mM glycine and 0.2%
gelatine)(9) . The colloidal protein A-10 nm gold suspension
obtained from Aurion (Wageningen, The Netherlands) was diluted 1:20
with the same buffer. BBMV were consecutively incubated with dilute IgG
for 90 min and protein A-10 nm gold for 60 min, then washed twice each
with blocking buffer, PBS, and distilled water and negatively stained
with 1% uranyl acetate. The bar represents 0.1
µm.
Fig. 1illustrates that various lipid exchange
proteins are liberated from BBMV in the absence of externally added
proteinases. Control experiments show that one or several steps in the
preparation of BBMV, but also subjecting BBMV to freeze-thaw cycles can
be responsible for the observed release of membrane proteins. In light
of these findings two questions arise, 1) are some or all of these
water-soluble lipid exchange proteins interrelated and/or related to
the integral membrane protein responsible for cholesterol and/or PtdCho
absorption in BBM(); 2) in case they are not related to this
protein what are their origin and physiological role? The first
question is pertinent to the isolation and purification of the
cholesterol receptor of the BBM. The major problem of isolating
integral proteins from BBMV is that detergent solubilization of BBM is
accompanied by the activation of intrinsic proteinases resulting in
massive degradation of membrane proteins and total loss of enzymatic
activities(24) . It was shown that the activity of the
intrinsic proteinases is difficult to control with proteinase
inhibitors and various mixtures thereof(24) . If the
water-soluble lipid exchange proteins described here were indeed
related to the cholesterol receptor of the BBM, the purification of
these proteins would be desirable. The usefulness of antibodies raised
against these proteins is obvious: these antibodies would be employed
in immunoaffinity chromatography for the isolation and purification of
the cholesterol receptor of the BBM.
In an attempt to shed light on the first question, the 13-kDa protein was purified to homogeneity and polyclonal antibodies were raised against this protein. It is the smallest water-soluble lipid exchange protein least contaminated as judged by SDS-PAGE(2) . The purification by cation exchange chromatography on Mono S HR 5/5 described here is easier, faster, and significantly cheaper than affinity high pressure liquid chromatography on Nucleosil-phosphatidylcholine described in a previous publication (2) and therefore better suitable as a routine method for purifying this protein.
The lipid exchange protein present in peak
1, which has an apparent molecular mass of 100 kDa(2) ,
and the 13-kDa protein both catalyze the exchange of PtdCho,
phosphatidylinositol, and cholesterol (Fig. 1A)
exhibiting features of nonspecific lipid transfer
proteins(25) . In contrast, the lipid exchange protein present
in peak 2 with an apparent molecular mass of 22 kDa mediates PtdCho and
phosphatidylinositol exchange but not cholesterol exchange. The 13-kDa
protein is not only functionally related to the 100-kDa lipid exchange
protein, but also immunologically: the polyclonal antibody against the
13-kDa protein cross-reacts with the 100-kDa protein, but not with the
22-kDa protein of peak 2 and the polyclonal antibody against the
100-120-kDa protein of peak 1 cross-reacts with the 13-kDa
protein (cf. Fig. 4, A-C). The peak 2 protein
appears to be functionally and immunologically different from the 13-
and 100-kDa lipid exchange proteins.
The relations between the three
water-soluble lipid exchange proteins are further corroborated by their
lipid binding properties as studied by ESR spin labeling. The 100- and
13-kDa protein bind both spin-labeled cholestane and PtdCho indicating
that their lipid binding site have properties reminiscent of
nonspecific lipid transfer proteins (25) . Formation of a
complex between these proteins and either 3-doxyl-5-cholestane or
5-doxyl-PtdCho is indicated by a strong immobilization of the spin
label as evident from the large hyperfine splittings observed (cf. Fig. 6and Table 1). The splittings are
significantly larger than those observed when the spin labels are
present in lipid bilayers or micelles (Table 1). This behavior is
contrasted by that of the 22-kDa lipid exchange protein which under
comparable experimental conditions shows no binding of
3-doxyl-5
-cholestane. From the comparison of the functional,
immunological, and lipid binding properties of the three water-soluble
lipid exchange proteins we conclude that the 13-kDa protein is related
to the 100-kDa protein and the properties of these two proteins are
different from those of the 22-kDa lipid exchange protein.
The
origin of the strong 13-kDa band in the immunoblot analysis of BBMV (Fig. 3A) is unclear although a similar observation was
reported previously(26) . The 13-kDa protein of the BBM could
be either a membrane-bound (membrane-associated) isoform of the 13-kDa
protein or it could originate from a larger integral membrane protein
by proteolytic cleavage. A third possibility would be that it is
identical to cytosolic sterol carrier protein 2 which is absorbed to
the BBM and subsequently slowly released. The close resemblance in
terms of physicochemical properties between the 13-kDa protein and
sterol carrier protein 2, particularly the high homology of the
NH-terminal amino acid sequence of these two proteins
support this possibility.
The results in Fig. 3would be explicable if the 13-kDa antigen were contaminated with proteins of higher molecular mass giving rise to a heterogeneous immune response. However, SDS-15% PAGE of the purified antigen clearly shows (Fig. 2) that this is not the case although, sometimes, due to the tendency of this protein to aggregate, dimers were detected. Hence the immunoblot results in Fig. 3indicate that the antibody against the 13-kDa protein cross-reacts with BBM proteins of apparent molecular mass greater than 13 kDa. Epitopes present on the 13-kDa protein are presumably also present on brush border membrane proteins of higher molecular mass. This notion is corroborated by using affinity-purified antibodies in the immunoblot analysis in Fig. 3. The results of Fig. 3may be rationalized in terms of membrane-bound isoforms of the 13-kDa protein. In this context results reported recently by Baum et al.(27) are relevant. These authors showed also by immunoblot analysis that polyclonal antibodies raised against rat liver sterol carrier protein 2 detect several high-molecular weight isoforms of this protein in tissue homogenates of rat liver and rat small intestines. Cross-reactions were observed with hepatic proteins of 14, 25, 27, 30, 45, 58, and 120 kDa and with small intestinal proteins of 14 and 30 kDa and a weak one with a 58-kDa protein. Several papers have appeared over the last few years providing convincing evidence for the presence of high-molecular weight isoforms of nonspecific lipid transfer proteins in different cell and cell organelles (see for instance, (11) and (28) -31). Alternatively, the immunoblot analysis of Fig. 3could be rationalized by postulating that the proteins that cross-react with the polyclonal antibody against the 13-kDa protein are derived proteolytically from one large integral BBM protein by the action of intrinsic proteinases. This notion is supported by the observation of similar band patterns in the immunoblot analysis of supernatant proteins and BBMV (cf. Fig. 3). In this case the water-soluble lipid exchange proteins including the 13-kDa protein could be part of a large integral BBM protein from which they are cleaved off proteolytically. We know that intrinsic proteinases are activated during the preparation of BBMV and it is likely that these proteinases are also activated by dissolving BBMV in SDS for PAGE.
The partial inhibition shown in Fig. 5is consistent with a previous report showing that the same antibody partially inhibits cholesterol absorption from mixed taurocholate micelles(32) . Unless the observed inhibition is an unspecific effect, it suggests that the 13-kDa protein is related to the integral membrane protein responsible for cholesterol/PtdCho absorption, i.e. to the cholesterol receptor of the BBM. The time lag observed may be due to the antigenic site being initially inaccessible to the antibody. The integral membrane protein may have to undergo a conformational change prior to the binding of the antibody. Immunogold staining (Fig. 7) lends support to this interpretation. BBMV as used here are oriented right side out (33, 38) and are impermeable to proteins of the size of trypsin(24, 34) . For positive immunogold labeling to be observed as shown in Fig. 7a, BBMV have to be incubated with the antibody for more than 30 min. The immunogold labeling clearly shows that the antibody interacts with a protein exposed on the external or luminal side of the BBM. In this context the results of the papain treatment of rabbit small intestinal BBMV are pertinent(35) . By using actin as a marker for proteins located on the cytosolic side of the BBM, papain was shown to cleave only proteins exposed on the external or luminal side of the BBM and to have no access to the cytosolic side(36) . Papain treatment of BBMV under standard conditions (35) liberates about 65% of the total membrane protein. The loss in protein is accompanied by a dramatic reduction in activity of enzymes known to be exposed on the external (luminal) side of BBM. For instance, the activities of aminopeptidase and sucrase isomaltase were shown to drop to levels of 25 and 18% of their original activity, respectively(35) . Concomitantly the protein-mediated absorption of cholesterol from mixed bile salt micelles is significantly reduced and the absorption of PtdCho from egg PtdCho SUV is practically abolished(1, 2, 3) . After papain treatment of BBMV, immunogold labeling was also greatly reduced. Immunogold labeling and papain digestion of BBM taken together provide therefore good evidence that the cholesterol receptor is exposed on the external or luminal side of the BBM. These experiments also support the notion that the 13-kDa protein is related to the cholesterol receptor.
The behavior of BBMV prepared from human small intestines and supernatant proteins produced from these vesicles merits discussion. The human BBMV closely resembled rabbit small intestinal BBMV in their lipid absorption properties. Both cholesterol and PC absorption by human BBMV are protein-mediated analogous to rabbit small intestinal BBMV reported before(1, 2, 3) . The rate constants are also comparable to those measured with rabbit BBMV. What was said for rabbit small intestinal BBMV concerning the reaction order and mechanism of cholesterol absorption is also valid for human BBMV. After proteolytic treatment of human BBMV with papain or proteinase K, cholesterol absorption became a passive diffusion process with half-times on the order of 10 h (data not shown).
The analogous behavior of the two kinds of BBMV is extended to the production of supernatant proteins. As evident from Fig. 1B, water-soluble lipid exchange proteins of very similar molecular mass and lipid exchange properties are liberated from human BBM by freeze-thaw cycles. Human peak 3 proteins mediated cholesterol and PC exchange between two populations of SUV and this lipid exchange was totally inhibited by the anti-rabbit 13-kDa antibody (data not shown). The inhibition was identical to that shown in Fig. 4C in which rabbit peak 3 protein was used as catalyst(32) . The strong immunological cross-reactivity between the anti-rabbit antibody and the human 13-kDa protein points to a high homology between the rabbit and the human 13-kDa protein, indicating that this class of proteins is highly conserved. The high homology observed in this class of lipid exchange proteins may account for the generally pour quality of the antibodies raised against these lipid exchange proteins. This manifests itself in weak or lacking immune responses (cf. Fig. 4, 5, and 7). For instance, with some antibodies raised against the 13-kDa protein no effect on cholesterol and PC absorption by BBMV was observed.
It is clear that the work presented does not provide an answer to the two questions raised at the outset of the discussion. At the best it may provide a working hypothesis. Taken all experimental evidence together we propose as a working hypothesis that the 13- and 100-kDa proteins are proteolytic products of the cholesterol receptor of the BBM. Evidence in favor of this proposal comes from 1) the similarity in functional, imunological, and lipid binding properties of the 13- and 100-kDa lipid exchange proteins; 2) the partial inhibition of lipid absorption by BBM in the presence of the anti-13-kDa antibody; 3) the positive immunogold labeling of BBMV using this antibody together with the papain treatment of BBMV; and 4) the immunoblot analysis of BBMV and supernatant proteins yielding similar band patterns.
The 22-kDa lipid exchange protein is both functionally and immunologically different from the other two water-soluble lipid exchange proteins and very likely of a different origin. Clearly more work is required to better characterize it and to clarify its relationship to other lipid exchange proteins.
In order to test our working hypothesis we are currently purifying the 100-kDa lipid exchange protein. By raising antibodies against this protein we should stand a good chance of elucidating the interrelationship between the water-soluble lipid exchange proteins liberated from the BBM and also answering the important question of whether or not the 100-kDa protein is related to the cholesterol receptor.