The apo-lipoproteins of human plasma high density lipoprotein: A study of their lipid binding capacity and interaction with lipid monolayers

BIOCHIMICA

THE

ET BIOPHYSICA

APO-LIPOPROTEINS

LIPOPROTEIN: INTERACTION

GERM-IN

CAMEJO,

OF HUMAN

A STUDY WITH

‘55

ACTA

OF THEIR

LIPID

ZADILA

April 9th.

HIGH

BINDING

DENSITY CAPACITY

AND

MONOLAYERS

M. SUAREZ

Centro de Biofisica y Bioquimica, Institute Apavtado 1827, Cavacas (Venezuela) (Received

PLASMA LIPID

AXIJ VICTOR

MUmOZ

Venezolano de Investigaciones

Cientijicas (I. V.I.C.),

1970)

SUMMARY I. The lipid-free protein moiety (apo high density lipoprotein) of human plasma high density lipoprotein (d 1.063-1.210) was fractionated by preparative polyacrylamide gel electrophoresis in the presence of 8 M urea into five components.

Fractions b and c, the major components, were shown to be responsible for the high surface activity (surface denaturation) and the lipid monolayer penetration of the whole apo high density lipoprotein. 2. Phospholipids dissolved in hexane are transferred by apo high density lipoprotein and Fractions b and c to the aqueous phase where the lipids are found firmly bound to the proteins. Apo high density lipoprotein bound more phospholipids than Fractions a, b or c. Tritium exchange experiments indicate that after the binding of phospholipids there is an increase in the exposure of peptide regions of apo high density lipoprotein to the solvent. The results are discussed with regard to the possible structure of high density lipoprotein and its capacity to interact in vivo and in vitro with lipids at interfaces.

INTRODUCTION

The plasma high density lipoprotein (d 1.063-1.210) appears to have important functions concerning the transport and exchange of lipids. It has been shown that high density lipoprotein activates in vitro the action of the enzyme lipoprotein lipasel. Lossow et aLa, on the other hand, found that the enzyme lecithin : cholesterol acyltransferase is closely associated with high density lipoprotein and GLOMSET~ has presented evidence which suggests that the interaction between this enzyme and high density lipoprotein could be one of the mechanisms used for the exchange of lipids between circulating lipoproteins and cell membranes. To understand the details of the process of exchange and transport of lipids in which high density lipoprotein seems to be involved, knowledge of the structure and physico-chemical properties of this lipoprotein and its components is required. The isolation of the protein moiety of high density lipoprotein as a soluble material by SCANU~ opened the way to B&him.

Biophys.

Acta, 218 (1970) 155-166

G. CAMEJO et Ul.

156

studies that established some of the singular properties of this apolipoprotein (apo high density lipoprotein). Apo high density lipoprotein was shown to be capable of binding to micellar suspensions of phospholipidsl and also to lipids dissolved in petroleum ether5. CAMEJO et ah6 demonstrated that rat apo high density lipoprotein had a high surface activity and a large capacity to penetrate lipid monolayers when compared with other proteins. These results were confirmed and extended by COLACICCO~.These studies suggest that the structure of apo high density lipoprotein is designed for interactions at polar-non-polar interphases. Apo high density lipoprotein was thought to be made of a protein with a molecular weight of approx. 25000 (refs. I, 4, 6-S). However, recently SHORE AND SHORES and SCANU et aLlo demonstrated that the apo high density lipoprotein is made of several proteins which could be separated by chromatography on DEAE-cellulose9 and gel filtration10 using buffers with 8 M urea as eluants. The heterogeneity of apo high density lipoprotein prompted several questions: (I) are all the components capable of binding lipids ?, (2) is there any specificity in their lipid binding capacity?, (3) do all components share the surface activity of the whole apo high density lipoprotein? The results reported

in this paper deal with the answers to some of these questions.

METHODS Preparation of plasma high density lijoprotein and a$o high density lipoprotein Blood from fasting young donors was used as a source of plasma. High density lipoprotein (d 1.063-1.210) was isolated as previously described”, with one additional step; after dialysis, the isolated high density lipoprotein was passed through a Sephadex G-zoo Column (Pharmacia-Fine Chemicals, Uppsala, Sweden) of 1.8 cm x 45 cm, in order to eliminate any contaminating albumin. In all steps during the separation of high density lipoprotein, EDTA (sodium salt) was present at 0.5 mM concentration. The proteins from high density lipoprotein were delipidated with ethanol-acetone”. The lipid-free apo high density lipoprotein was dissolved in water adjusted to pH 10.5 with NH,OH, lyophilized and stored at -15” until used. Preparation and isolation of high density lipoprotein and rat liver lipids The ethanol-acetone extract of high density lipoprotein was evaporated to dryness at reduced pressure. The residue was dissolved in chloroform-methanol (2 : I, by vol.) and washed according to the procedure of FOLCH at a1.12. The lipids dissolved in the chloroform phase, as well as the other lipid solutions used were stored at -15” in amber glass bottles fitted with teflon-lined screw-caps. Rat liver lipids were also extracted and washed as in the method of FOLCH et a1.l2. Individual phospholipids were isolated from high density lipoprotein total lipids by preparative thin-layer chromatography and subsequent elution from the silica gel by methanol-chloroform (2 : I, by vol.) and chloroform. The homogeneity of the fractions isolated was checked by one- and two-dimensional thin-layer chromatography. The identity of the fractions was established by comparing their chromatographic behaviour with that of commercially obtained standards (Applied Science Lab. Inc., Pa., U.S.A.). Polyacrylamide gel electrophoresis of ape high density lipoprotein A vertical cell was used (EC Apparatus Corp., Pa., U.S.A.). B&him.

Biophys.

Acta,

218

(1970)

155-166

The gel was 8%

LIPID

BINDING

BY PLASMA

HIGH

DENSITY

I57

LIPOPROTEIN

cyanogun (EC Apparatus Corp.) prepared in 0.5 M Tris-HCl (pH 9.0) and made 8 M with respect to urea. The electrode chambers were filled with 0.04 M Trisglycine (pH 8.3). Usually, 375 V were applied for at 20 f 2’ was circulated through the cooling plates Preparative gel electrophoresis was performed but the gel was 8 mm thick and 15-20 mg of apo applied in a single slot along the gel.

150 min. During the run, water of the cell. using the same cell and buffers high density lipoprotein were

The analytical gels were stained either with Coomassie brillant blueI or with amido black. The results were evaluated by densitometry, using a linear transport (Gilford Instruments, Oberlin, Ohio, U.S.A.), and light of 600 nm wavelength. The different protein bands in the preparative gels were localized by scanning a nonstained gel slab using quartz cuvettes and light of 280 nm wavelength. After identifying the segments of the gel containing the ultraviolet-absorbing bands, the slabs were cut and placed in a cell for electrophoretic elution similar to that designed by GORDON’~. After adding IO ml of 50 mM Tris-glycine buffer (pH 8.4) to each compartment of the cell containing the gel slabs, current (5 mA) was passed through the cell for 12 h. At the end of this period the buffer was recovered from the compartments and the procedure repeated twice with ro-ml portions of buffer. The combined buffer portions of each compartment were concentrated by ultrafiltration using a Diaflo membrane and pressure cell (Amicon Corp. Lexington, Mass., U.S.A.). Urea and salts were eliminated by passage of the concentrated fractions through a column of Bio-gel P-2 (Bio-Rad Lab., Richmond, Calif., U.S.A.). The appearing in the void volume were lyophilized and stored at -15” Swface

pressure

protein fractions until used.

measurements

The procedure applied has been described previouslysy7 but the modifications were introduced: the circular trough was completely made and water at 25 f r” was circulated around the trough. The buffer was 5 HCI (pH 7.40). The water used to prepare all solutions was demineralized

following of teflon mM Trisand twice

distilled over alkaline permanganate in an all-glass still. A detailed discussion of the methods used for measuring surface activity (surface denaturation) of proteins and penetration of lipid monolayers has been presented by COLACICCO~. In the surface activity measurements and in those of monolayer of protein in all cases was 2 pg/ml of sub-phase.

penetration,

the final concentration

binding by ape high density lipoprotein and its components The lipids were dissolved in re-distilled hexane. The proteins were dissolved in 5 mM Tris-HCl buffer (pH 7.40) containing 0.15 M NaCl. The aqueous phase containing the protein and the hexane phase containing the lipids were placed in test tubes (7.5 cm x 1.7 cm) with teflon-lined screw-caps. The volume ratio of the Lipid

two phases was I to I, and from 0.25 to I mg of protein was incubated with 0.5-2 mg of lipids. The tubes were placed into a rack fitted to a wrist-action shaker (Burrel, Model CC, Pittsbourgh, Pa., U.S.A.) in such a way that the axis of rotation for all tubes was the same. The tubes were shaken for 12 h at 22’ &_ 2’. At the end of this period the tubes were centrifuged at 1550 rev/min for 10-15 min and the hexane phase withdrawn. A a-ml portion of clean hexane was layered on top of the aqueous phase and after manual inversion of the tube this was centrifuged again and the Biochim.

Biopkys.

.4&a.

218 (1970)

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15s

G. CAMEJo et cd.

hexane combined with the first portion. This procedure was repeated twice. For lipid analysis of the hexane phase, aliquots were used directly. When the aqueous phase was going to be analyzed for lipids it was first extracted and washed according to the procedure of FOLCH et a1.l2 and portions of the extract used. For studies of gel filtration behaviour, tritium exchange, and electron microscopy the aqueous phases were used as obtained after the final hexane washing. No evidence of protein denaturation was detected in the aqueous phase. Tritium exchange ex$eriments In general, the procedure of ENGLANDER’~ was followed, except that a IO cm x 1.8 cm column of Rio-gel P-4 was used in place of the 6 cm x 3 cm column of Sephadex G-25 described in the method. The P-4 column had a faster flow and resolved completely the tritium-labeled protein and the tritiated water. To I mg of the protein dissolved in I ml of 0.5 mM Tris-HCl buffer (pH 7.40) was added 0.2 ml of tritiated water containing activity equivalent to I mC, and the exchange was allowed to take place for 6 h at 22’ & 2’. Analytical methods Protein was measured by the procedure of LOWRY et a1.16. Phospholipids were of digested samples. Cholesterol was estimated by phosphorus determinationI determined by the method of BOW&IAN AND WOLFI”. The fatty acid composition of phospholipids was obtained by gas-liquid chromatography of the fatty acid methyl esters prepared by treatment of the lipids with 60/b(by vol.) H,SO, in methanol. Electron nzicroscopy Samples containing o.r-0.50/6 proteins were visualized by means of negative contrast using 2% sodium silica tungstate adjusted to pH 7.6. The formvar-coated grids were examined at 72000 times magnification obtained by a Siemens Elmiskop IA electron

microscope.

RESULTS Fractionation of ape high density lipoprotein Analytical or preparative polyacrylamide electrophoresis in gels containing 8 M urea resolved the lipid-free moiety of high density lipoprotein into six bands (Fig. I). Electrophoretic elution of these bands yields the two major components with little contamination. Fig. 2 represents the densitometric pattern of an analytical polyacrylamide gel electrophoresis of the isolated major components. Fraction a (Fig. I), upon re-electrophoresis, appeared to be contaminated with about 30% of Fractions b and c. Fraction d was obtained with no detectable contaminants. The two peaks which constitute Fraction e were not possible to be separated. Protein recovery in the preparative system was 60-75% of the protein applied to the gel. The electrophoretic pattern of apo high density lipoprotein in gels containing 8 M urea was neither modified by prolonged (up to 7 days) incubation of apo high density lipoprotein in 8 M urea nor when the apo high density lipoprotein was treated with I mM N-ethylmaleimide prior to the incubation with urea. Re-electrophoresis of the isolated Fractions b, c, d and e in gels containing 8 M urea did not Riochiwz. Biophys. .?cta,ZIH (7970) I.js-166

LIPID

BINDING

BY PLASMA

HIGH

DENSITY

LIPOPROTEIN

Fig. 1. Scanning of a preparative electrophoresis of apo high acrylamide gel containing 8 M urea. The gel was not stained.

‘59

density

lipoprotein

in 80/, poly-

Fig. 2. Patterns obtained from analytical electrophoresis in 8% polyacrylamide gels prepared in 8 M urea. The two peaks shown correspond to Fractions b and c obtained from apo high density lipoprotein by preparative runs. The gels were stained with Coomasie brilliant blue.

show other bands besides those of the material loaded in the gel. These results suggest that the bands observed are distinct proteins and not different aggregation states of the same protein. Nearly identical patterns were obtained with apo high density lipoprotein prepared separately from the plasma of six different donors. Surface

activity of a$o high density li$oprotein and its components Fractions b and c, the major apo high density lipoprotein components, show a high surface activity when injected into the sub-phase. The rate at which they raise to the interfase and the final surface pressure observed with the whole apo high density lipoprotein

reached were similar to that (Fig. 3). Fraction a formed a

Fig. 3, Surface activity of apo high density lipoprotein (aHDL) and its isolated fractions, temp. 25”. The subphase in all cases was stirred by means of a teflon-coated magnetic bar turned by means of an external bar attached to a synchronous motor. The experimental points are an average of two or three determinations. The protein concentration in the sub-phase was 2 pg/ml. B&him.

Biophys.

Acta,

218 (1970)

155-166

160

G. CAMEJO

film at a lower rate than Fractions b or c, but since it was contaminated 30:/o of Fractions b and c it was not possible to determine whether

ft al.

with about the surface

activity was a property of Fraction a or of the contaminants. Fractions d and e exhibit a much lower capacity to form films than Fractions b, c or the apo high density lipoprotein. We must therefore conclude that the high surface activity observed in the apo high density lipoprotein ponents which possess this property. Interaction

of ape high density lipoprotein

is caused by the presence

of two com-

with lipid monolayers

Films of high density lipoprotein total lifiids. When Fractions b, c, d and e of apo high density lipoprotein are injected below monolayers made of high density lipoprotein total lipids, (percentage composition : phospholipids, 52.2 ; cholesterol esters, 38.5; triglicerides, 2.9; cholesterol, 6.4) at an initial pressure of z dynes/cm, clear differences are observed (Fig. 4). Fraction b shows the fastest increase in surface (I)

Fig. 1. Penetration lipids high density temp. 25’.

by apo high density lipoprotein lipoprotein, initial film pressure

fractions of monolayers prepared from total dynes/cm, protein concentration 2 /Lg/ml,

2

and a higher final pressure. Fraction c, although it reaches almost the same final pressure as Fraction b, shows a slower rate. Fraction e, which shows a smaller surface activity than Fraction d was, however, capable of increasing the film pressure to 14.2 dynes/cm compared with 11.5 for Fraction d. As in the surface activity experiments, Fractions d and e reach much lower final pressures than Fractions b and c. Whole apo high density lipoprotein gave a curve that followed that of Fraction b. (2) Neutral lipid jilms. The final pressures reached by these films (percentage composition: cholesterol esters, 80.5; triglicerides, 6.1; cholesterol 13.4) after injection of the apo high density lipoprotein fractions were: 20.2, 17.5, 12.0 and 10.5 dynes/cm for Fractions b, c, e and d, respectively. The initial pressure of the film in all cases was 2 dynes/cm. (3) Phosphatidyl choline films. This phospholipid was selected because it is the Biochzm. Biophys.

Acta,

218 (1970)

155-166

LIPID

BINDING

BY PLASMA HIGH DENSITY

major polar constituent

161

LIPOPROTEIN

of the high density lipoprotein

lipids. Penetration

experiments

were conducted to a film initial pressure of 16 dynes/cm. This pressure clearly discriminates the film penetration capacity of Fractions b and c and that of Fractions d and e. Fractions d and e did not penetrate phosphatidyl choline films at 16 dynes/ cm but whole apo high density lipoprotein and Fractions b and c were still capable of penetrating the monolayer (Fig. 5).

Fig. 5. Penetration by apo high density lipoprotein (aHDL) and Fractions b and c of monolayers prepared from high density lipoprotein phosphatidyl-choline high density lipoprotein, initial surface pressure 16 dynes/cm, protein concentration 2 bug/ml, temp. ~5~.

Li$id

bindilzg by ape high density lipoprotein and its components Apo high density lipoprotein binds more phospholipids than Fractions a, b or c (Table I). Fractions d and e did not bind detectable amounts of lipids. In this incubation procedure neither apo high density lipoprotein nor the isolated fractions bound cholesterol, cholesterol esters, or triglycerides to any appreciable extent. TABLE

I

Composition OF HIGH DENSITY LIPOPROTEIN PHOSPNOI,IPIDS BOUND BY HIGH DENSITY LIPOPROTEIN AND ITS MAJOR PROTEIN FRACTIONS In all cases I mg of total lipids extracted from high density lipoprotein were dissolved in 0.5 ml of hexanc and shaken with 0.50 mg of each of the proteins dissolved in 0.5 ml of buffer. The percentage composition of the phospholipids present in the hexane solution was : lysophosphatidyl choline, 6.0 : sphingomyelin, z I .o ; phosphatidyl choline, 66.9 ; phosphatidyl ethanolamine, 6. I. Pvotein

Apo high density lipoprotein Trypsin-treated apo high density liprotein * * * Fraction a Fraction b Fraction c Serum albumin

T/, Composition

of the bou+zd phospholipids

wag Phospholipid bound pev wg protein

Sphingmnyelin

Phosphatidyl choline

I.0 -0.83*

21.3**

72.9

5.8

17.2 ‘7.9 16.1

72.4 72.8 76.6

10.3 9.3 7.3

Phosphatidyl ethanolamine

0

0.68-0.45 0.64-0.40 0.68-0.50 0

* Maximum and minimum value obtained in four independent determinations. * * Average of three independent determinations. * * * To a apo high density lipoprotein solution containing I mg/ml of protein was added trypsin (IO pg/mg apo high density lipoprotein) 2 h before the incubation with the hexane solution of lipids. Biochim.

Biophys.

Acta,

215 (1970) 155-166

G. CAMEJO

162

et al.

Trypsin-treated apo high density lipoprotein, serum albumin, as Fractions d and e, failed to transfer phospholipids from the hexane solution to the aqueous phase. That in the aqueous phase, the apo high density lipoprotein and its fractions were bound to the lipids is shown by the results of gel-filtration experiments. Apo high density lipoprotein and Fractions a, b and c penetrated Sephadex G-zoo gel, but once incubated with the lipids the proteins were found in a different peak containing the phospholipids. The results from electron microscopic studies of the products obtained after incubation with the lipids indicate also that the protein is firmly bound to the lipids. When observed with the electron microscope by negative contrast, high density lipoprotein is clearly seen as quasi-spherical particles of about 150 .Y_ (Fig. 6A). The lipid-free apo high density lipoprotein, however, is almost beyond the resolving power of the method (Fig. 6B), but the partially reconstituted lipoproteins can be observed as quasi-spherical structures with a size which is dependent upon the amount of phospholipids bound (Fig. 6C). These particles are not lipids in aqueous suspension, since aqueous suspension of lipids observed by negative contrast in the electron microscope are converted to lamellar and tubular structures, not spherical particles as the lipoproteins. The major fractions of apo high density lipoprotein bound a mixture of phospholipids with a percentage composition similar to that bound by apo high density lipoprotein. Apo high density lipoprotein and Fractions b and c when incubated separately with purified phospholipids, bound more phosphatidyl choline than phosphatidyl ethanolamine, but always the whole apo high density lipoprotein bound more phospholipid than the isolated fractions (Table II). Pure sphingomyelin

Phospholipids were isolated by thin-layer chromatography from a lipid extract obtained from high density lipoprotein. In all caSeS 0.25 rni: of protein clissolved in 0.5 ml of buffer was incubated with 0.5 mg of the phospholipid dissolved in 0.5 ml of hexane.

~~~ Protrin

mg Phosphatid_yl cholzm bound $w mg +votcin

mg Phosphatidyl rthanolamzne bound per mg pVot?ilZ

I. j%I.jh* 1.36-1.16

0.70~0.50 0.37-0.26

T.IL-1.00

0.37-0.34

Apo high density lipoprotein Fraction b Fraction c Strum albumin *

Maximum

0

and minimum

values

0

obtained

in four independent

determinations.

the other major phospholipid present in the lipids of high density lipoprotein, was not soluble in hexane and therefore it was not possible to assay its binding. To explore if the preferential binding was evident when incubated with other lipid mixtures, the apo high density lipoprotein was shaken with a hexane solution of rat liver total lipids. The results presented in Table III show that the composition of the bound lipids is not only dependent upon the affinity of apo high density lipoprotein for the different phospholipids but also upon the composition of the lipid mixture with which it is incubated. Some interesting observations from this experiment should be pointed out. The apo high density lipoprotein or its fractions did Biochirn.

Bioph>js. Acta,

218 (1970) 155-166

LIPID

BINDING

BY PLASMA

HIGH

DENSITY

LIPOPROTEIN

163

Fig. 6. Electron micrographs of plasma high-density lipoprotein (A), apo high density lipoprotein (B), and (C) partially w-constituted high density lipoprotein obtained by incubation of ape high density lipoprotein with high density lipoprotein total lipids, 0.7 mg of phospholipid bound per mg of protein. The arrows in (A) indicate the high density lipoprotein particles with an apparent beaded sub-structure. This beaded appearance can also be observed in (C). In apo high density lipoprotein (B) no spherical particles of the size of high density lipoprotein were seen, only the thread-like structures indicated by arrows were observed. This structures were not detected in control grids containing buffer and silicotungstate. All preparations visualized by negative contrast with zq/o sili~ot~ngstat~ (pH 7.6). Total magnification 405000 x . The segment indicates zoo A. B&him.

Riophys.

Acta, 218

(1970)

155-166

G. CAMEJO t?,fal.

164 TABLE

III

RIKDINGOF RATLIVER PHOSPIIOLIPIDS

BY

HIGH

DENSITY

LIPOPROTEIK

lipid extract obtained irom rat Iiver was used as source of the phospholip~ds. 2 mg of rat liver lipids dissolved in I ml of hexsne mere incubated with 0.50 mg of apo high density lipoprotein dissolved in 0.5 ml of buffer. _... .__._ ._~~ __. .-.. ~~ _.. ~ -_ .--.. ~~ Phospholipids _ _ __~ .-.. _____ Sphingofla~elin ___~ _--. LYSOPhosphntidyl PhosphatPhosphatidyl idylimsitol ( ?) ethmolamine phosphatidyl CfaOliPIP chottwc X

__-

y. Composition

of rat

liver phospholipids O’ Composition of rat IfQer phospholipids bound by ape high density lipoprotein

/” Composition of high density lipoproteiff phospholipids

.__~~

_-

._-_. .._._ ~~_..

-

~_~

6.8”

6.2

60.7

1.3

22.”

9.0**

17.6

48.2

0

24.5

Cl.o*

21.0

6G.g

0

0’

6.1

._.

Average results from duplicate determinations. * * Average results from duplicate determinations

*

not bind

a phospholipid,

tentatively

in three different

identified

incubations.

as phosphatidyl

inositol

and only

whole apo high density lipoprotein and Fraction a bound Iysolecithin. To explore if the high density lipoproteins had any preference for phosphatides with some fatty acid composition, the proteins were incubated with egg phosphatidyl choline and phosphatidyl ethanolamine. The fatty acid composition of the phospholipids bound and that of the ones remaining in the hexane phase was analyzed, no differences were observed. However, when incubated with synthetic dipalmitoyl choline none of the proteins bound this lipid, indicating acids is a requirement for binding.

that

unsaturation

of some of the fatty

Tritiwm exchange experiments The tritium exchange experiments suggest appreciable conformational differences between the apo high density Iipoprotein and the partially reconstituted lipoproteins. The specific activity of apo high density lipoproiein after 6 h of exchange was x1331 counts/min per mg protein (& 541, SE.) compared with 14560 counts/ bound to min per mg protein (& 673, SE.) f or the apo high density lipoprotein phosphatidyl choline. This preparation contained 0.9 mg of phosphatidyl choline per mg of proteins. Since aqueous suspensions of phosphatidyl choline did not exchange tritium in the conditions used here, the increase in specific activity of the partially reconstituted lipoprotein has to be ascribed to protein-bound tritium. DISCUSSION

The functional roles in which the plasma high-density lipoprotein appears to be involved suggest that its apo high density lipoprotein is designed to facilitate the transport of lipids between biological interfases l+,S,ls. This protein is capable of penetrating lipid films at surface pressures at which other proteins do not6*’ and can transfer lipids from a non-polar environment to an aqueous phase5. The properties are probably connected since we can envisage that some of the steps in the process

LIPID BINDING

BY PLASMA

of lipid transfer collection

from

HIGH

DENSITY

a hydrophobic

of the apo high density

LIPOPROTEIN

I65

to a hydrophylic

lipoprotein

environment

at the interfase;

are:

(I) the

(2) penetration

of the

lipid film present at the interface; (3) binding of appropriate segments of the polypeptide to the polar regions on the film; (4) and finally, the transfer ( f the formed lipoproteins to the bulk of the aqueous phase. The failure of apo high density lipoprotein to transfer in vitro triglycerides and cholesterol esters to the aqueous phase, as described by SODHI AND GOULD~ and confirmed in the present work, can be explained along the above lines. The lipids collected at the hexane-water interfase should be only those with polar regions, the neutral lipids probably are not accessible to the apo high density lipoprotein. In vivo apo high density lipoprotein is associated to neutral lipids, therefore, in the biological interfase at which the protein binds lipids, both polar and non-polar lipids must be exposed to the protein. SHORE AND SHOREY, and SCANU et aLlo demonstrated that apo high density lipoprotein is a mixture of polypeptides. The two major components of apo high density lipoprotein isolated by preparative polyacrylamide electrophoresis appear

equivalent to Fractions III and IV isolated by gel filtration from high density lipoprotein (d 1.063 and 1.125) by SCANU et al. lo. SCANU et a1.2ohave shown that these proteins can bind sonicated suspensions of high density lipoproteins (d 1.063 and 1.125) total lipids. These two major components of apo high density lipoprotein, Fractions b and c, are the cause of the high surface activity and capacity to penetrate lipid monolayers shown by the high density lipoprotein. Although no large difference between apo high density lipoprotein and Fractions b and c were evident from monolayer studies, the lipid-binding experiments showed differences between these preparations. Apo high density lipoprotein bound more lipids than the isolated Fractions a, b, or c indicating some co-operative process between the components of apo high density lipoprotein. The failure of Fractions d and e to bind lipids and their lower surface activity suggest that they should serve functions related with lipid binding in the high density lipoprotein complex.

not directly

No clear specific interaction between a lipid and an apo high density lipoprotein fraction appears to exist. Apo high density lipoprotein as well as Fractions b and c bind more phosphatidyl choline than ethanolamine, a finding that may explain the low concentration of this phospholipid in high density lipoprotein. The experiments with rat liver lipids show, however, that the proportions in which the lipids are bound to apo high density lipoprotein are not only a function of the partial specificity of the proteins but also of the proportion in which the lipids are present in the hydrophobic phase. Apo high density lipoprotein was not capable of binding dipalmitoyl phosphatidyl choline, a result that suggests some discrimination of the protein regarding the non-polar region of the lipid besides the one observed towards the polar region of the phospholipids. Because most of the phospholipids found in plasma and membranes contain both saturated and unsaturated fatty acids, the significance of this discrimination is not clear at the moment. The controlled partial re-constitution of lipoproteins offers the possibility of studying the effect which lipid binding has on the conformation of the proteinzl. Results from our laboratory indicate that when bound to lipids the protein from high density lipoprotein is in the surface of the particle in a conformation in which its peptide backbone is at least as exposed to the aqueous environment as in the lipid-free protein 11. The tritium exchange experiments suggest that when lipid-bound, B&him.

Biophys.

Acta,

218

(1970) 155~~(0

I66

G. CAMEJO

et al.

the high density lipoproteins have 22(f4, more exchangeable hydrogens than the lipid-free apo high density lipoprotein. This result suggests a model for high density lipoprotein in which these particles are visualized as quasi-spherical structures with a mosaic surface made of the polar heads of lipids and the protein, with most of this last component in contact with the environment. Such a model places protein where it could facilitate the interactions between lipid components biological interfases.

tire of

ACKNOWLEDGMENTS

We wish to express

our gratitude

to Mr. J.

Aristimurio

for his valuable

as-

sistance during the electron microscop!, studies, and to Dr. Karl Gaede for helpful advice during the preparation of the manuscript. We also thank Mr. R. Pingarron and Mr. J. L. Rigorra for the photographic labor and Miss Arlette Duprc for secretarial work.

1 A. Sc.mc, J. Hzol. Chew,

IO II 12 13 14 15 16

242 (1967) 7”. J, Lossow, S. N. SHAH APED I. L. CHAIKOFF, Lliochm. Biopjhycys. Acta, I 16 (1966) 172. A. GLonm?T, ,I. Lipid RPS., 9 (1968) 155. SCAP~U, ,J. Lipid Res., 7 (1966) 295. S. SOOIII AND K. GORDOX GOULD, J. Biol. CIwm.. 242 (‘967) 1205. CAMEJO, G. COLACICCO AND RI. M. RAPPORT, ,I.Lipzd Res., 9 (19%) ~(2. COLACICCO, ,J. Colloid Iztcvfacr Sci., 29 (1969) 345, CAMEJO, fhochrmh’y, 6 (1967) 3228. R. SHORE A.uI)\'.SHORE, Rzochrmistvy, H (1969) 4510. :\. SCANU, J. I‘OTH, c. I
17 18 19 20

J. M. BEVERIDCE AxU S. E. JOHNSOK, Can.]. I&s., 27 (1949) 1959. Ii. I?.BOWMAS AK;D R. C. \X'OLI', Clin. Clzem., 8 (1964) 126. 1'.p\'. SCHUMAKER AND G. H. I\DAMS, ilm. Iicv.Biochem.. 38 (1969) 689. STILLER AND L. .L\LBERS,Biochemistry, A. Scmu, 13. CUMP, J. TOTH, S. KoGA, I?..

2 W. 3 J, 1 A. 5 H. 6 C. 7 C;. 8 G.

9

265.

1327. 21 21. SCAKV AKD Ii. HIRZ, Pvoc. Hiochim.

Uqbhys.

Acta,

218 (1970)

Xati. .4cad. Sci. C..‘., 155-166

59 (1968)

890.

9 (r97oj