High density lipoprotein conversion mediated by human plasma phospholipid transfer protein.

Phospholipid transfer protein (PLTP) was purified from lipoprotein-free human plasma, obtained upon treatment of plasma with dextran sulfate and Ca2+, by employing a series of column chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified PLTP showed a single main band, corresponding to the molecular mass of 78 kDa. However, isoelectric focusing of the purified preparation gave multiple bands with pI ranging from 4.3 to 5.1, indicative of microheterogeneity. Purified PLTP was shown to possess not only phospholipid transfer activity, but also high density lipoprotein (HDL) conversion activity (Tu, A.-Y., Nishida, H. I., and Nishida, T. (1990), FASEB J. 4, A2148; Jauhiainen, M., Metso, J., Pahlman, R., Blomqvist, S., van Tol, A., and Ehnholm, C. (1993) J. Biol. Chem. 268, 4032-4036). Isolated HDL3 was enlarged to the size of HDL2b upon incubation with purified PLTP for 6 h at 37 degrees C at the PLTP/HDL3 molar ratio of approximately 1:45. Both the HDL conversion and the phosphatidylcholine transfer activities of purified PLTP were effectively inhibited by rabbit anti-PLTP immunoglobulin G. The primary importance of PLTP in the HDL enlargement that occurs in human plasma upon incubation at 37 degrees C was shown by the strong inhibitory effect of the anti-PLTP immunoglobulin G. The process of PLTP-mediated HDL enlargement was accompanied by the release of apoproteins, primarily apoA-I. HDL3 enlargement mediated by PLTP was effectively inhibited by the addition of free fatty acids.

well as, phospholipids (PL) among plasma lipoproteins, and is often called cholesteryl ester transfer protein or CETP (1-3). Phospholipid transfer protein (PLTP) facilitates the transfer of only PL, but not the neutral lipids (4-6). It may exert a physiological role in the transfer of phospholipids from the surface coat of lipolyzed chylomicrons and very low density lipoproteins to HDL (7,8).
Although plasma was previously shown to contain conversion factor activities transforming HDL into larger and smaller particles (9, lo), the activities were more recently attributed to the effect of LTP (11, 12). Our study, however, revealed that the conversion factor activity responsible for the HDL enlargement was exerted by PLTP (13). The same conclusion was recently reported by Jauhiainen et al. (14). In both studies (13, 14), PLTP purified to apparent homogeneity was shown to convert HDL primarily to larger particles. Jauhiainen et al. (14) also showed the absence of inhibition of the HDL conversion by monoclonal anti-LTP antibody, thus indicating an independence of the conversion to LTP. In this communication, the direct proof for the involvement of PLTP for the HDL enlargement was provided by the inhibition of both PC transfer and HDL conversion activities of purified PLTP preparations and of human plasma by the anti-PLTP IgG prepared. Although the addition of fatty acid is known to enhance the LTP-mediated conversion of HDL primarily to smaller particles (12, 15), it showed an inhibitory effect on PLTP-mediated HDL enlargement.

Buffer
Experiments were performed in 39 mM phosphate buffer containing 60 mM NaCl (ionic strength 0.16, pH 7.4) unless otherwise specified.
The term EDTA designates 0.025% solution unless its concentration is given.
Preparation of Labeled PC Vesicles and PC Cholesterol Vesicles Single bilayer PC vesicles or PC-cholesterol vesicles were prepared according to the method of Batzri and Korn (19) as previously described (18). An ethanol solution (0.3 ml) containing 12.5 pmol of egg PC, [3H]dipalmitoyl PC (I3H]DPPC) (3.7 pCi), and 0, 2.1, 4.2, or 6.2 pmol of cholesterol was rapidly injected into 4.7 ml of phosphate buffer while stirring under N2. After incubation of the mixture for 30 min at room temperature, the ethanol used for lipid injection was removed by exhaustive dialysis at 4 "C against phosphate buffer. The amounts of cholesterol present in the preparations of PC-cholesterol vesicles corresponded to 0, 14, 25, and 33 mol % of the total lipids. These vesicles were stored at 4 "C and used within 1 week after the preparation.

Labeling of LDL
The labeling of LDL was carried out essentially according to the method of Albers et al. (6). An ethanol solution (0.3 ml) containing [3H]DPPC (3.5 pCi) was injected into 4.7 ml of freshly isolated LDL preparation in phosphate buffer. The mixtures were incubated at 37 "C for 3 h and then dialyzed exhaustively at 4 "C against the same buffer. The preparations used in experiments were stored at 4 "C and used within 2 weeks of the preparation. The methods used for the determination of lipid and protein compositions of LDL were previously described (20,21).

Determination of PLTP Activity
The PC vesicles/HDL3 assay system was primarily used for the determination of PLTP activity. The assay mixtures consisted of 35 pl of PC vesicles (88 nmol of egg PC and 3.9 nmol of I3H]DPPC with the specific activity of 32.9 pCi/pmol) as PC donors, HDL3 (98 nmol of PL) as the acceptor and varying amounts of PLTP in phosphate buffer containing 60 mM NaCl with a final volume of 280 p1. The assay mixtures were incubated at 37 "C for 15 min. After incubation, the vesicles in the assay mixtures were precipitated by adding 80 pl of 0.0085% dextran sulfate and 40 pl of 0.2 M MgC1, (21). After standing for 20 min in an ice bath, the mixtures were centrifuged at 2,900 X g for 20 min to obtain the supernatant solution containing HDL3. Then an aliquot of the supernatant (200 pl) was analyzed for its radioactivity. The PC transfer activity was computed by subtracting the blank values, which included the spontaneous transfer, from the total PC transfer obtained in the presence of PLTP.
Inhibition of the transfer activities of plasma PLTP and purified PLTP by rabbit anti-PLTP IgG was determined by using both the PC vesicles/HDLs assay system and LDL/HDL assay system. For the LDL/HDL assay system, the donors, I3H]DPPC-LDL (51 nmol of PL), and acceptors, HDL2 (255 nmol of PL), were incubated with various amounts of PLTP samples in phosphate buffer at 37 "C for 30 min with a final volume of 300 ul. After the incubation, LDL in the assay system was precipitated by adding 45 pl of 0.02% dextran sulfate and 30 pl of 60 mM MgC& (21). After standing at 4 "C for 20 min, the mixtures were centrifuged at 2,900 X g for 20 min to precipitate radiolabeled LDL. An aliquot of the supernatant (150 pl) containing HDL was measured for radioactivity. The PL transfer from LDL to HDL2 was calculated by subtracting the transfer value in the absence of PLTP from the total amounts of [3H]DPPC transferred in the presence of PLTP.

Purification Procedures
All steps were carried out at 4 'C or in an ice bath and all solutions used were precooled in a refrigerator. Chromatographic runs were monitored at 280 nm with the ISCO model UA-4 or 5 absorbance monitor (ISCO, Lincoln, NE). The centrifugations were carried out with a Du Pont/Sorvall RC-5 refrigerated centrifuge. Dialysis was carried out by using Spectra/Por regenerated cellulose membranes (Spectrum Medical Industries, Inc., Houston, TX) unless otherwise noted.
Step 1: Dextran Sulfate Treatment-This step is similar to that used for the purification of lecithin-cholesterol acyltransferase (22) and LTP (23). To 1 liter of the stirred plasma, 1.5 liter of glassdistilled water and 500 ml of dialyzed dextran sulfate (10 g) were added. This was followed by the addition of 4 M CaC12 to the final concentration of 0.1 M. The mixture was stirred gently for 15 min and then centrifuged at 16,000 X g for 20 min in order to remove the insoluble dextran sulfate-lipoprotein complex.
Step 2: Phenyl-Sephnrose Chromatography-This chromatography was carried out in a similar manner as described for the purification of LTP and lecithin-cholesterol acyltransferase (17, 23). To the stirred dextran sulfate supernatant fraction, solid NaCl was added to increase the ionic strength to 0.8. This mixture was applied to a phenyl-Sepharose CL-4B column (3.5 X 21 cm), which was previously equilibrated with 0.8 M NaCl containing EDTA. The column was washed with 1,300 ml of the NaCl solution and with 800 ml of 39 mM phosphate buffer containing EDTA. After elution of lecithin-cholesterol acyltransferase and LTP with 600 ml of 2.9 mM phosphate buffer (pH 7.4) containing EDTA (18, 24), the PLTP fraction was eluted with 500 ml of 25% ethanol.
Step 3. CM-Cellulose Chromatography-An aliquot of 2 M acetate buffer (pH 4.5) was added to the 25% ethanol fraction to yield 50 mM buffer concentration. The fraction was applied to a CM-52 column (3.6 x 16 cm), which was equilibrated with 50 mM acetate buffer. The column was washed with 300 ml of the acetate buffer containing 50 mM NaC1, and the PLTP fraction was eluted with 700 ml of 50 mM acetate buffer containing 100 mM NaCl.
Step 4: DEAE-Cellulose Chromatography-The CM-cellulose fraction was dialyzed against 4 mM phosphate buffer (pH 7.4) containing EDTA by using Spectrum hollow fiber bundles (132292) and was applied to a DE-52 column (1.9 X 9 cm) equilibrated with the phosphate buffer. The column was washed with 150 ml of the phosphate buffer containing 50 mM NaCl and EDTA. The PLTP adsorbed to the column was then eluted with 150 ml of the phosphate buffer containing 150 mM NaCl and EDTA.
Step 5: Heparin-Sephurose Chromatography-The DEAE-cellulose fraction was dialyzed against 4 mM phosphate buffer containing EDTA (pH 7.4) and was applied to the heparin-Sepharose CL-4B column (1.5 X 6 cm) equilibrated with 4 mM phosphate buffer containing EDTA. The column was washed with 240 ml of 4 mM phosphate buffer containing 100 mM NaCl and with 80 ml of phosphate buffer containing 200 mM NaCl. The PLTP fraction was eluted with 80 ml of phosphate buffer containing 400 mM NaCl and EDTA and then dialyzed against 4 mM sodium phosphate buffer containing 150 mM NaCl (pH 6.8).
Step 6. Hydroxylapatite Chromatography-The dialyzed Heparin-Sepharose fraction was applied to a hydroxylapatite column (1.9 X 2.5 cm), which was previously equilibrated with 4 mM sodium phosphate buffer (pH 6.8) containing 150 mM NaCI. The column was washed with 35 ml of the equilibration buffer. PLTP was eluted with 40 ml of 25 mM sodium phosphate buffer containing 150 mM NaCl. The PLTP fraction was dialyzed against 0.4 mM phosphate buffer (pH 7.4) containing EDTA and 2 mM NaN3. When concentrated preparations were required, an Amicon Centriprep-10 Concentrator was used to concentrate the dialyzed fraction. Occasional contaminants in the hydroxylapatite fraction were removed by gel permeation chromatography using Superose 12 column (1 X 30 cm) and 39 mM phosphate buffer, pH 7.4, containing EDTA and 2 mM NaN3 as the equilibration and elution buffer.
Gradient Gel Electrophoresis (GGE) GGE was carried out essentially according to the method of Blanche et al. (24) by using Pharmacia 4-30% and Bio-Rad 4-20% non-denaturing polyacrylamide gradient gel. Stained gel was scanned with an ISCO model 1312 gel scanner and analyzed by using high molecular weight electrophoresis calibration standards (Pharmacia).

Preparation of Rabbit Anti-Human PLTP IgG
Approximately 150 pg of purified human plasma PLTP in 1.0 ml of 4 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl were mixed with the same volume of Freund's complete adjuvant. The oil suspension, 2 ml per rabbit (body weight of approximately 2.4 kg), was injected into multiple intradermal sites (23). The second injection of a similar amount of PLTP in 2 ml of oil suspension (Freund's incomplete adjuvant: 0.15 M NaC1, 1:1, v/v) was given 2 weeks after the first injection. The blood was collected from the ear vein 1 week after the second injection. IgG fraction against human plasma PLTP was purified from the serum by using dextran sulfate/CaCl, treatment, ammonium sulfate precipitation, and DEAE-cellulose column chromatography. The control IgG was isolated from non-immunized rabbit serum following the same procedures (23).
The specificity of anti-PLTP IgG was determined by a solid phase enzyme-linked immunosorbent assay method using goat anti-rabbit IgG conjugated with alkaline phosphatase and Bio-Rad alkaline phosphatase substrate kit according to the procedure given by the manufacturer. The color that developed was measured at 405 nm. A standard curve was constructed for 0-18 ng of purified PLTP ad-sorbed to microtitration plates in order to approximate the reactivities of various apoproteins, lecithin-cholesterol acyltransferase, and LTP, that were adsorbed to the plates, toward the anti-PLTP IgG. Assay methods with the use of antibody adsorbed to the plates were not feasible because of a relatively low titer of our anti-PLTP IgG preparation. The enzyme-linked immunosorbent assay indicated that the anti-PLTP IgG is highly specific for PLTP. Purified apoA-I, apoA-11, apoC-111, and apoE adsorbed to the plates showed no reactivity toward the anti-PLTP IgG. Purified apoD, lecithin-cholesterol acyltransferase, and LTP gave the reactivities equivalent to 0.5, 2.2, and 1.7%, respectively, of that of purified PLTP on protein weight basis. These proteins, however, showed no significant inhibitory effect on the binding of anti-PLTP IgG to the adsorbed purified PLTP. Anti-PLTP IgG obtained did not inhibit significantly the cholesteryl ester formation by purified lecithin-cholesterol acyltransferase (22) and cholesteryl ester transfer reaction by purified LTP (23).

Analytical Methods
The protein concentration was determined by the Lowry method (25) using crystalline bovine serum albumin as the standard. The enzymatic and fluorimetric method of Gamble et al. (26) was modified for the determination of small amounts of free cholesterol and cholesteryl ester. The amounts of PL were determined by the method of Bartlett (27). Triglyceride contents were measured by an enzymatic assay using an enzymatic assay kit from Sigma. SDS-PAGE on a 10% polyacrylamide gel was performed using a Mini-Protean I1 Dual Slab Cell (Bio-Rad) as previously described (23). The molecular weight was determined by comparing with those of the standard proteins as previously described (23). Isoelectric focusing was carried out on a 5% polyacrylamide gel by using a Mini-Protean I1 Dual Slab Cell (Bio-Rad) as previously described (23). The pH gradient was determined by using the standard proteins with known isoelectric point (Pharmacia).

RESULTS
Purification of PLTP from Human Plasma-The purification of PLTP from 1 liter of human plasma is summarized in Table I. Dextran sulfate/Ca2+ treatment, under the conditions given, allowed the effective precipitation of all plasma lipoproteins (22, 23). This lipoprotein removal from plasma was necessary for its application to the phenyl-Sepharose column, although no increase in the specific activity was achieved. In phenyl-Sepharose column chromatography, water has been commonly used for the elution of PLTP (5, 6, 28). We observed that the substitution of 25% ethanol for water doubled the yield of PLTP activity eluted. Prior to the elution of PLTP, the column was eluted with 2.9 mM phosphate buffer in order to obtain a fraction containing LTP and lecithincholesterol acyltransferase (17, 23). This elution also reduced the contaminant protein in the PLTP fraction. Unlike CMcellulose chromatography, DEAE-cellulose chromatography did not lead to a substantial increase in the specific activity.
This step, however, was necessary to remove contaminant proteins which, otherwise, would be present in trace amounts in the final PLTP preparations.
Heparin-Sepharose served as an excellent affinity chromatography column for PLTP (6,28,29). Elution of PLTP with the least amount of contaminant proteins was obtained by increasing the NaCl concentration of 4 mM phosphate buffer (pH 7.4) from 200 to 400 mM. The last purification step, hydroxylapatite column chromatography, was very effective in removing remaining contaminant proteins. The final purification of PLTP was approximately 38,700-fold over the starting plasma PC transfer activity. Since about 10% of the PC transfer activity of the starting plasma was contributed by LTP under the assay conditions used, the final purification fold could be 10% higher than the value given. The hydroxylapatite fraction exhibited a single major band by SDS-PAGE and by staining with silver stain reagent (Fig. 1, Panel  A ) . From the mobility on the polyacrylamide gel, an apparent molecular mass of 78 kDa was computed.
In spite of the apparent homogeneity exhibited by SDS-PAGE, the purified PLTP showed multiple bands upon isoelectric focusing on 5% polyacrylamide gel. The isoelectric points of the band ranged from 4.3 to 5.1 (Fig. 1, Panel B ) .
Nearly identical patterns consisting of at least 10 bands were obtained for three different purified preparations. The presence of both 0.2% Triton X-100 and 2 M urea in the gel was essential to resolve the multiple bands. The multiple bands of LTP due to its microheterogeneity were previously resolved in the presence of Triton X-100, but without addition of urea (23), and those of lecithin-cholesterol acyltransferase were resolved in the absence of both Triton X-100 and urea (30).
Immunoinhibitwn of the PC Transfer Actiuities of Purified PLTP and Plasma PLTP--Rabbit anti-PLTP was obtained from the rabbit which had received an intradermal injection of purified PLTP. When anti-PLTP IgG was added to purified PLTP, almost complete inhibition of PC transfer activity was observed with both PC vesicle/HDLa and LDL/HDL assay systems (Fig. 2, Panel A, curues 2 and 3). The addition of control IgG did not significantly inhibit the PC transfer activity (curue I ) . When purified rabbit anti-PLTP IgG was added to plasma samples, approximately 90% of total PC transfer activity of plasma was removed at high IgG concentrations (BOO Kg) with the vesicles/HDL3 assay system (Panel B, curue 3). However, when the LDL/HDL assay system was used, the inhibitory effect of anti-PLTP IgG was reduced; about 40% of the transfer activity remained in the plasma (curue 2). In both assay systems, the transfer activities remaining in the plasma were due to the PC transfer activity of LTP present in the plasma (6). The differences in the inhib- The PC transfer activity of plasma was assayed by using 2 pl of human plasma. 'The total PC transfer activity includes the transfer activities of not only PLTP but also of LTP. The contribution of LTP to both starting plasma and dextran sulfate/CaClz fraction was approximately 10%. low PI standard proteins (Pharmacia) ( l a n e 2 ) were carried out on a 5% polyacrylamide gel containing 0.2% Triton X-100,2.0 M urea, and Ampholine (pH 4-6) for 5 h a t 0.6 watt. After treatment with 10% trichloroacetic acid overnight, the gel was also stained with silver stain reagent. The PLTP sample applied contained approximately 2 pg of purified P L T P in 0.2% Triton X-100, 2 M urea, and 12% glycerol. control IgG and anti-PLTP IgG were added to purified P L T P (32 units) or 5 pl of plasma. The mixtures were preincubated in 175 pl of phosphate buffer containing EDTA for 1 h a t 4 "C, and then centrifuged a t 7,000 rpm for 30 min prior to the assay incubation. Curue Z in each panel represents the transfer activities determined with both LDL/HDL2 and PC vesicles/HDLs assay systems after preincubation with control IgG. Curves 2 and 3 show the activities obtained with LDL/HDLz and PC vesicles/HDLs assay systems, respectively, after preincubation with anti-PLTP IgG. The transfer activity is given as a percentage of the activities relative to that obtained in the absence of antibody. The activities (expressed as nanomoles of PC transfer per h) of purified PLTP in the absence of antibody, determined with vesicle/HDLs and LDL/HDL, assay systems, were 31 and 4.6, respectively, while those of plasma P L T P activities were 26 and 4.5 nmol, respectively. itory effects of anti-PLTP IgG on the plasma PLTP in the two assay systems were due to considerably higher PC transfer activity of P L T P with the vesicles/HDL3 assay system than with the LDL/HDL* assay system (legend for Fig. 2). We observed that PC transfer activities of LTP purified to homogeneity (23) in both assay systems were comparable. It appeared that vesicles served as very efficient PC donors for PLTP but not for LTP.
Effect of Cholesterol on the P C Transfer Activity-To determine if our purified PLTP exhibits properties identical with those of partially purified PLTP fractions previously used by other investigators (31, 32), the PC transfer activities were determined by using vesicles containing 0 (curve 1 ), 14 (curve 2), 25 (curve 3 ) , and 33 (curve 4 ) mol % of cholesterol as PC donors (Fig. 3). The progressive increase in the cholesterol content resulted in a marked decrease in the PC transfer activities which were plotted as a function of PLTP concentration. The percent PC transfer, as a function of mol % cholesterol in vesicles (Fig. 3, inset) was obtained in the presence of 40 ng of PLTP in comparison to the activity in the absence of cholesterol. When cholesterol content of the vesicles was increased to 33 mol %, the transfer rate was reduced to only about 20% of the transfer activity obtained in the absence of cholesterol. These results are indeed in agreement with the observations made by other laboratories using partially purified P L T P (31, 32). After the incubation, LDL in the assay mixtures was precipitated by adding dextran sulfate and MgCl, to the final concentration of 0.01% and 6 mM, respectively. After standing for 20 min in ice, the assay mixtures were centrifuged at 2,900 X g for 20 min, and the supernatant solutions containing vesicles were carefully and completely removed. The LDL precipitate was dissolved in 100 p1 of 0.1 N NaOH, and an 80-pl aliquot was analyzed for radioactivity. The PLTP-mediated PC transfer activity was calculated by subtracting the blank values, which included the nonfacilitated transfer, from the total PC transfer obtained in the presence of PLTP. In the inset, the percentage of the PC transfer activity in the presence of 40 ng of PLTP, relative to the activity in the absence of cholesterol, was plotted as a function of cholesterol mol ' 70 in the donor vesicles.
concentrations on the extent of HDL enlargement was studied by incubating isolated HDL, with varying amounts of purified PLTP at 37 "C for 6 h and by determining the GGE patterns. Upon addition of 400 ng of purified PLTP (equivalent to PLTP/HDL, molar ratio of approximately 1:45), HDL3 was converted to a higher molecular weight species having a Stokes' diameter of 10.7 nm (Fig. 4, pattern 3 ) , which corresponds to the average size of HDLZb (24). Heterogeneous patterns were obtained especially in the presence of low concentrations of PLTP (33 ng, pattern 7; 50 ng, pattern 6 ) , possibly reflecting the differences in susceptibility of various HDL3 subspecies to PLTP-mediated enlargement. This heterogeneity, however, was progressively reduced by increasing  It is noteworthy that even in the presence of the lowest amount of PLTP (PLTP/HDL3 molar ratio of approximately 1540, pattern 7), the enlargement of HDL, was detected by the appearance of the leading peak having the diameter of approximately 9.0 nm. The Stokes' diameter of the leading peak, representing the enlarged particles, was plotted as a function of PLTP/HDL, molar ratio (Fig. 5). The enlarged peak approached the maximal size at the molar ratio of approximately 0.011 (1:90).
To show changes in the density and sizes of HDL, particles that occur by treating isolated HDL3 with PLTP, four fractions (d < 1.125, 1.125 < d < 1.21, 1.21 < d < 1.25, and d > 1.25 g/ml) were obtained by sequential ultracentrifugation of HDLs samples (1.48 mg as protein) incubated with 50,000 units (8.1 pg) of purified PLTP. The recovered HDL fractions were analyzed by gradient gel electrophoresis and the recovery of HDL3 proteins and lipids were determined. The distribution of control HDLs-protein, incubated without PLTP, in the four fractions, was 8, 76, 9, and 7%, respectively, while that of HDL3-protein, incubated with PLTP, in the corresponding four fractions was 30, 37, 4, and 29%, respectively. It was apparent that control HDL, gave the recovery of the HDL, primarily at 1.125 < d < 1.21 g/ml with a small amount of the lipoprotein recovered at d < 1.125 g/ml. Upon incubation of HDL, with PLTP (PLTP/HDL, molar ratio of 1:150), the recovery of the lipoprotein at 1.125 < d < 1.21 ml was greatly reduced with a major portion of the lipoprotein recovered at d < 1.125 g/ml. Furthermore, the size of lipoprotein particles in both fractions were substantially larger than the corresponding control samples (data not shown). The conversion of HDL, into less dense, larger particles was accompanied by the release of substantial amounts of apoproteins; 29% of HDL, protein was recovered from the d > 1.25 g/ml fraction when HDL3 was incubated with PLTP. Analysis of the protein by SDS-PAGE showed that apoA-I was a major apoprotein released during the conversion. Control HDL3 incubated in the absence of PLTP gave the recovery of approximately 7% of the protein (mostly apoA-I) from the d > 1.25 g/ml fraction. This may reflect the release of relatively small amounts of apoproteins known to occur upon recentrifugation of HDL (33).
Apparently the change in the density and size of HDL3 upon incubation with PLTP was due to the apoprotein removal from HDL,, resulting in the conversion of apoprotein deficient HDL3 particles into the lighter and larger HDL3 and HDLzlike particles.

Immunoinhibition of HDL Conversion Activity-We ob-
served that the addition of excess anti-PLTP IgG to the incubation mixtures containing isolated HDL3 and PLTP prevented the PLTP-mediated HDL enlargement. In order to determine the contribution of human plasma PLTP to the HDL enlargement that occurs upon incubation of plasma at 37 "C, plasma samples treated with varying amounts (0-1000 pg) of anti-PLTP IgG were incubated for 8 h at 37 "C. In the absence of anti-PLTP IgG, the plasma incubation caused the enlargement of HDL, as previously observed (10,34). As compared to the HDL, in control plasma samples kept at 4 "C ( Fig. 6, patterns 1 and 8 in both A and B ) , the incubation for 8 h at 37 "C resulted in the formation of enlarged HDL (patterns 2 and 7). The addition of increasing amounts of anti-PLTP IgG to plasma progressively reduced the extent of HDL enlargement (patterns 3 and 4). The enlargement was almost completely prevented at the highest concentration of anti-PLTP IgG (pattern 5 ) . The addition of non-immunized IgG (pattern 6 ) did not reduce the extent of HDL enlargement. We also observed that the treatment of plasma with anti-LTP IgG (23) and with anti-lecithin-cholesterol acyltransferase IgG prepared against purified lecithin-cholesterol acyltransferase (18) did not show any appreciable inhibitory effect on the enlargement.
Effect of Fatty Acid on HDL Conversion-To study the effect of fatty acid on the PLTP mediated HDL3 conversion, HDL3 preparations, preincubated with varying amounts of palmitic acid, were incubated with PLTP at 37 "C for 6 h.
The HDL3 enlarged by PLTP in the absence of palmitic acid, gave a Stokes' diameter of 10.3 nm (Fig. 7, pattern 3 ) . This enlargement was progressively inhibited upon increasing the amount of palmitic acid added to HDL3 (patterns [4][5][6][7][8]. In the presence of 1.5 and 3 pg of palmitic acid, the GGE pattern of the incubation mixtures showed considerable heterogeneity (patterns 4 and 5), possibly reflecting the differences in the affinities of HDL subspecies for palmitic acid. The extent of heterogeneity in the GGE patterns was reduced with increasing amounts of palmitic acid added to HDL3 (patterns [6][7][8]. The inclusion of 8 pg of palmitic acid nullified the HDL enlargement (pattern 8).

DISCUSSION
In the present study, human plasma PLTP was purified and some basic factors influencing the PLTP-mediated HDL conversion process were defined. The HDL conversion was observed as an enlargement of HDL3 into HDLz-like particles. The presence of HDL conversion factor activity in plasma was first observed by Gambert et al. (10). The incubation of plasma or isolated HDL in the presence of the d > 1.25 g/ml fraction of plasma at 37 "C for 24 h caused a decrease in small HDL and an increase in large HDL. Rye and Barter (9) also showed the presence of the conversion factor. The incubation of HDL with the partially purified conversion factor led to the formation of larger and smaller HDL particles (9). The identity of HDL conversion factor (11) remained unknown and it was later concluded that the HDL conversion was indeed caused by LTP (12). We showed that PLTP is responsible for the enlargement of HDL (13). Jauhiainen et al. (14) also reached the same conclusion. By using anti-PLTP IgG, we provided direct evidence that PLTP is indeed responsible for the enlargement of isolated HDL as well as the enlargement of HDL in the plasma system. PC transfer activity of purified PLTP was completely abolished upon pretreatment of the assay mixtures with anti-PLTP IgG. We recently used discoidal bilayer particles containing PC, cholesterol, and apoA-I as model lipoproteins for the PLTP-mediated enlargement (35). The enlargement was effectively inhibited when the mixtures containing the discoidal bilayer particles and purified PLTP were pretreated with anti-PLTP IgG in a similar manner as given in the legend for Fig. 6. Since the apoA-I used for preparation of discoidal bilayer particles did not cross-react with anti-PLTP IgG, the inhibition must have been due to the immunoprecipitation of PLTP in the mixtures. No other protein was present in the mixtures. This observation as well as the high specificity of anti-PLTP IgG used in the present study indicated the primary involvement of PLTP in the HDL enlargement and not other factors or co-factors.
The possible physiological importance of PLTP-mediated HDL enlargement was indicated upon comparison of the HDL patterns of control plasma (Fig. 6, patterns 1 and 8 in both A  and B ) and those of the plasma incubated at 37 'C for 8 h (patterns 2 and 7). Although the incubation caused a rather limited increase in the Stoke's diameter of the main peaks, the appearance of shoulders with larger Stoke's diameter suggested the presence of HDL subspecies which were rapidly converted to HDLz-size particles. Near complete prevention of the HDL enlargement by excess anti-PLTP IgG (pattern 5) suggested the possible involvement of PLTP in the HDL enlargement in vivo.
Based on the stoichiometry of the HDL, conversion (Fig.  5), PLTP apparently served as a catalyst or an enhancer of the process. The lowest PLTP/HDLa molar ratio used (1540, pattern 7) roughly corresponds to the physiological molar ratio assuming the plasma PLTP concentration of 2 mg/liter. At the molar ratio of 1:360 (pattern 6 ) , a major portion of HDL3 was converted to HDLz,-size particles (Stoke's diameter of 9.3 nm) upon incubation for 6 h at 37 "C. Although at the PLTP/HDL, molar ratio of 1:45 (pattern 3), HDL3 was almost completely converted to HDLzb-size particles, physiological relevance of the conversion at such a high molar ratio is questionable. This conversion, however, indicated the near upper limit for the size of the particles (Fig. 5) that can be produced from HDL3.
The PLTP-mediated HDL conversion was accompanied by the release of apoproteins, primarily apoA-I in accordance with previous observations (9, 14,36). It is interesting to note that when isolated HDL was treated with 3 M guanidine HCl (37) or heated to 70 "C (38), the apparent HDL size increased, which seemed to be correlated with the dissociation of apoA-I. Apparently, the amount of apoA-I in HDL particles regulated the size of the particles. The preferential release of apoA-I upon treatment of HDL3 with PLTP may be the result of the weaker affinity of apoA-I for HDL, (16, 39). It is well known that HDL is extremely heterogeneous with respect to apoprotein and lipid compositions (40-42). The role of PLTP in the apoprotein release needs to be investigated with refined model systems and various HDL subfractions, such as HDL containing apoA-I without apoA-I1 and HDL containing both apoA-I and apoA-I1 (42).
Although pretreatment of HDLa with palmitic acid was shown to inhibit the enlargement of HDL,, plasma free fatty acids are not likely to exert inhibitory effects on the HDL enlargement in vivo. Free fatty acid concentrations in plasma are very low and yet most free fatty acids are in association with albumin, thus making unbound free fatty acid concentrations extremely low (43). The effect of palmitic acid was studied in order to compare to the effect shown by fatty acids on the LTP-mediated HDL conversion (12,15). It appears that PLTP and LTP give opposing effects on the HDL size transformation. Unlike PLTP, LTP is primarily responsible for the formation of smaller particles (11,12). It is possible that LTP induced HDL conversions to smaller particles are enhanced by incorporation of free fatty acid by expanding the HDL surface layer. While PLTP induced HDL enlargement is prevented by the surface layer expansion which interferes with the transient formation of surface coat deficient HDL particles needed for the enlargement.
The present study suggests a number of areas which should be addressed by future investigations. Many factors influencing HDL conversion as well as the mechanism of HDL enlargement need to be clarified. Although in the plasma, PLTP is primarily responsible for the enlargement of HDL, minor but significant roles of lecithin-cholesterol acyltransferase and LTP in HDL enlargement were indicated in our prelim-inary studies using model lipoprotein systems as well as plasma. The contributions of lecithin-cholesterol acyltransferase and LTP to HDL enhancement and maturation are to be defined under variety of conditions. Chemical characterization of PLTP is yet to be completed. N terminal amino acid of our purified PLTP appears to be blocked. Although freshly obtained purified PLTP showed at least ten bands with PI ranging from 4.3 to 5.1 on isoelectric focusing pattern, our preliminary study showed that upon extensive treatment of PLTP with Clostridiumperfringens neuraminidase (23,30), these bands converted into two intense bands with PI values between 7.5 and 8.0. The presence of PLTP isoforms as well as their microheterogeneity, due possibly to the differences in the number of sialic acid residues, are currently under investigation.