00l3-7227/91/1281-0547$02.00/0 Endocrinology Copyright© 1991 by The Endocrine Society

Vol. 128, No. 1 Printed in U.S.A.

The Thyroxine-Binding Site of Human ApolipoproteinA-I: Location in the N-Terminal Domain S. BENVENGA*, H. J. CAHNMANN, AND J. ROBBINS Clinical Endocrinology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

lipid-associated apoA-I, the pattern of hierarchy was variable, presumably related to the known markedly polydisperse nature of HDL, but a constant feature, in contrast to the case of isolated apoA-I, was that MAb 4 was more potent than MAb 14. Group II MAbs gave less than 3% inhibition in both isolated and lipidcomplexed apoA-I. Group III MAb 9 either failed to inhibit or gave 18-27% inhibition (one preparation each of HDL2 and HDL3). We conclude that the T4 site of apoA-I is in the Nterminal domain of apoA-I, closer to the epitope for MAb 16 than to that for MAb 18, and that conformational changes occurring when apoA-I is associated with lipids in the HDL particle alter the spatial relationship between some epitopes and the T4 site. Our definition of the T4 site of apoA-I is consistent with another set of data showing that heparin failed to inhibit [l25I]T4 binding to isolated apoA-I. Heparin is known to interact with clusters of basic residues, and these residues are concentrated in the midregion of apoA-I. (Endocrinology 128: 547552,1991)

ABSTRACT. We tested the ability of nine monoclonal antibodies (MAb) against human apolipoprotein-A-I (apoA-I), the 28.3-kDa major apoprotein of high density lipoproteins (HDL), to inhibit its photoaffinity labeling with [12SI]T4. Two forms were evaluated: isolated lipid-free apoA-I (Sigma or Calbiochem) and lipid-complexed apoA-I [HDL2, (density, 1.063-1.125 g/ml) and HDL3 (density, 1.125-1.210 g/ml)]. After labeling with 0.5 nM [mI]T« in the presence of MAb or normal mouse IgG, the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent densitometric quantitation of radioactivity associated with the 28.3-kDa band. Group I MAbs, namely those having epitopes in the N-terminal portion of apoA-I, include MAb 16 (epitopes at residues 1-16), 4 and 14 (residues 1-86), and 18 (residues 98-105); group II includes MAbs 7, 10, 15, and 17 (epitopes at residues 87-148); group III includes MAb 9 (residues 149-243). All group I MAbs inhibited [125I]T4 binding to isolated apoA-I with this order of potency: MAb 16 (-50% to -61%) > MAb 14 (-37% to -41%) > MAb 4 (-27% to -33%) > MAb 18 (-19% to -27%). In the case of

W

respective epitope within the primary structure of the protein) has been defined, and assuming the absence of allosteric effects, the inhibition by a given MAb of a given property of the protein indicates the region responsible for that property. We have recently used a panel of MAbs to localize the T4 sites of apoB-100, the structural apolipoprotein of low density lipoproteins (LDLs) (7). In this study we have evaluated the ability of nine MAbs against apoA-I to inhibit its interaction with [125I] T4, and we show that MAbs with N-terminal epitopes are the most effective inhibitors. Our conclusion that the T4 site of apoA-I is in the N-terminal third of the protein, in proximity to the epitope for MAb 16 (residues 1-16), is consistent with another set of data we gathered, namely the failure of heparin, a known inhibitor of T4 binding to the major T4 transport plasma proteins (8), to inhibit T4 binding to isolated apoA-I. Heparin binds to clusters of basic amino acids (Arg, Lys, His) (9), and the lowest concentration of these three amino acids is in the sequence 1-76.

E HAVE recently demonstrated that the major lipoprotein carriers of T4 are those belonging to the high density lipoprotein class (HDL) and that this binding reflects a specific interaction with their protein moiety, especially with apolipoprotein-A-I (apoA-I). This protein accounts for 50-60% of the total apoprotein mass and has a single site that binds T4 with moderate affinity (1). With the exception of transthyretin, for which the crystal structure is known (2), and some information on T4-binding globulin (TBG) (3), based mainly on its homology with ai-antitrypsin (4, 5), the position of the T4 site(s) of thyroid hormone-binding proteins is unknown. There is, however, an increasing use of monoclonal antibodies (MAbs) as tools to probe the physical and biological properties of proteins, including the apolipoproteins (6). Once the specificity (i.e. the position of the Received August 1, 1990. Address requests for reprints to: Dr. Jacob Robbins, Building 10, Room 8N315, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. * Visiting Associate, Fogarty Center. On leave from the Istituto di Clinica Medica I, Thyroid Unit, University of Messina School of Medicine, 98100 Messina, Italy.

Materials and Methods Proteins Electerophoretically pure lipid-free human apoA-I was obtained from Sigma (St. Louis, MO; lot 95F-9543) and Calbi547

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T4-BINDING SITE OF APOA-I

548

ochem (San Diego, CA; lot 507771) and was dissolved in 0.1 M Tris-HCl, pH 8.2. The two typical subclasses of HDL, HDL2 and HDL3, were isolated by ultracentrifugation (density, 1.0631.125 and 1.125-1.210 g/ml, respectively), dialyzed, and checked for the absence of contamination by LDL, human serum albumin, TBG, and transthyretin, as previously described (10). Three preparations of each subclass that were found to be contaminant free were used. Experiments with MAbs The nine MAbs tested, all of the immunoglobulin G (IgG) class and received as mouse ascites fluid, were a generous gift of Dr. L. Curtiss (La Jolla, CA), who has previously reported on their characterization (11-13). For the purpose of this study it is convenient to classify them into three groups: MAbs with N-terminal epitopes (residues 1-105; group I), which include MAb 4, 14, 16, and 18; MAbs with epitopes in the midportion of apoA-I (residues 87-148; group II), which include MAbs 7, 10, 15, and 17; and MAbs with C-terminal epitopes, which include MAb 9 (residues 149-243; group III). Figure 1 summarizes schematically the locations of these epitopes as well as those of the positively charged amino acids. The IgG fraction was isolated from the mouse ascites fluid by affinity chromatography (GammaBind G-Prepack column, Genex Corp., Gaithersburg, MD) according to the manufacturer's instructions. Normal IgG from nonimmunized mice (Calbiochem) was used as a control. Typically, the 100-^1 incubation mixture contained 5 ng apoA-I plus 1 jitg IgG adjusted to the final volume with 0.1 M Tris-HCl, pH 8.2, or 10 tig HDL plus 1 /xg IgG adjusted to the final volume with 0.1 M Tris-HCl, pH

Endo'1991 Vol 128* No 1

7.5-0.14 M NaCl-0.01% EDTA. In some experiments, mouse ascites fluid diluted 1:100 with 0.1 M Tris-HCl, pH 7.6, was used in place of IgG, in which case the control was buffer alone. After overnight incubation at 4 C in a 1.5-ml polypropylene microfuge tube (Sarstedt, Princeton, NJ), 0.5 nM inner ring labeled [125I]T4 (DuPont/New England Nuclear, Boston, MA; SA, 4400 Ci/mmol) was added, and the mixture was kept for a second overnight incubation at 4 C. Tubes were then irradiated at 23 C for 1 min with light above 300 nm (Corning filter 0-54, Medfield, MA; Osram HBO 100W/2 lamp, Bunton Instrument Co., Rockville, MD) at a distance of 21 cm from the A-215 lamp housing (Photochemical Research Associates, London, Ontario, Canada) (1, 14). The labeled products were precipitated with 5 vols cold acetone (14) and analyzed by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 16% or 18% acrylamide). Radioactivity associated with the 28.3-kDa band (apoA-I) was quantitated by laser densitometry. Experiments with heparin Inhibition of [125I]T4 (0.3 nM) binding to isolated apoA-I (100 Mg/ml) or HDL2 or HDL3 (both at -400 ng protein/ml) by 110 MM heparin (Sigma; therapeutic serum levels, ~2 ^M) was assessed by both photoaffinity labeling with subsequent SDSPAGE (conditions as above) and conventional equilibrium labeling with subsequent separation of bound from free radioactivity by gel filtration chromatography (Sephadex G-25 medium; Pharmacia, Piscataway, NJ) (1, 10). In either case incubation was performed at 23 C for 18-24 h. Quenching of tryptophan fluorescence

NH2

L

50

-100

T, binding domain

< 1•

MAb 16 1

243

200

COOH

Isolated apoA-I and one preparation of HDL2 were incubated for 24 h at 23 C with 10 ^M L-T4 dissolved in 0.04 N NaOH. Controls were incubated with 0.04 N NaOH alone. Trp fluorescence was measured with a Perkin-Elmer model MPF-66 spectrofluorometer (Norwalk, CT; excitation at 280 nm; emission at 337 nm).

J

Lipid binding domain

MAb 18 98 105

6

Group I

150

MAb 4,14 1

86 MAb 7,10,15,17

Group II

87

148 MAb 9

Group I I j

No. of basic residues/100 residues

Position of the tryptophans

149

Results

243

30-i

-30

20-

-20

10-

•

•

1 1 8

i 50

50

77 1 72

131

163

209

-10 -0 243

1 108

100

150

200

243

FlG. 1. Positions of the epitopes of the apoA-I monoclonal antibodies (MAbs), the 4 tryptophans, and the clustering of the 43 basic amino acids in human apoA-I. Note the correlation of the basic residue-poor N-terminal region of apoA-I (sequence 1-76) with the epitopes for MAbs 4, 14, and 16 and the basic residue-rich midportion of apoA-I (sequences 131-208 and, in particular, 131-162) with the epitopes for group II and III MAbs. The boundary between the T4-binding domain and the lipid-binding domain is placed at residue 99, the beginning of the tandem repeats of 22 amino acids forming the amphipathic ahelices, which have been proposed to be responsible for the interaction with lipids (18).

Experiments with MAbs The effects of MAbs on the photoaffinity labeling of both isolated and lipid-complexed apoA-I are summarized in Fig. 2, and representative autoradiographs are shown in Fig. 3. In the case of isolated apoA-I (Fig. 3A), except for a very faint 50-kDa band present in the IgGs (c/. Fig. 3B), the only labeled band was the apoprotein itself. In the case of the HDL preparations, additional radioactive bands were seen, especially a major band at the origin of the resolving gel. Some of these bands represent labeled T4-binding proteins in ascites fluid (Fig. 3C; cf. Fig. 3B), but others were also found in the absence of ascites fluid (Fig. 3C, control, and Fig. 3D). We assume that these latter may reflect [125I]T4 binding to apolipoproteins other than apoA-I and are currently

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T4-BINDING SITE OF APOA-I p - Form

ASCITES FLUID

MAb p-Group L* No.

I 14

16

n

7,10,15,17

19

14

16

18

7,10

JLI ,15,17

I

HDL, 60

30

?

20

^

10

iimiiiiiini

g 40 |

I

I

50

HDL-:

r-l

rTTMiiniiii

2

= I

=

!

A. Isolated apoA-I incubated with isolated IgG

FIG. 3. Representative SDS-PAGE autoradiographs of isolated or lipid-complexed apoA-I photoaffinity labeled with [128I]T4 in the absence or presence of group I MAbs to human apoA-I. The experiment shown for HDL2 is representative of those performed with mouse ascites fluid, while those for ApoA-I and HDL3 were performed with IgG. B, Photoaffinity labeling of isolated apoAI, MAb 14 ascites fluid, or MAb 14 IgG for reference. In A, C, and D, the extreme left lane shows labeling in the absence of MAb. The acrylamide concentration in A and B was 16%; in panels C and D, it was 18%.

18

ISOLATED IgG

m

70

Isolated ApoA-I

FIG. 2. Inhibition (as percentage of the control) of [125I]T4 photoaffinity labeling of isolated, HDL2-associated, or HDL3associated apoA-I by monoclonal antibodies against apoA-I. When three or •more experiments with the same protein and the same MAb were performed, the SD could be determined (indicated by error bars); most of the values given are the mean of two experiments. Since the four MAbs of group II gave uniformly negative results, these were combined. The shading of the bars identifies three preparations of HDL2 and HDL3. Note the agreement between experiments with ascites fluid and isolated IgG.

549

MAb

QJl

.$» 7

D.

C. HDL2 incubated with ascites fluid

—4141618 Resolving Gel

MAb Resolving Gel

apoA-I

apoA-I

investigating their nature. It is noteworthy, however, that neither the ascites fluid nor the isolated IgG contained 28-kDa proteins capable of binding T4 (Fig. 3B) that would have interfered with the 28-kDa band due to apoA-I. Both group II and III MAbs failed to inhibit [125I]T4 binding to isolated apoA-I (Fig. 2). Among the effective group I MAbs (19-61% inhibition) the order of potency was MAb 16 > 14 > 4 > 18. There was, therefore, a clear

4 141618

HDL3 incubated with isolated IgG MAb — 4 141618 Resolving Gel

apoA-I

apoA-I

NH2 —> COOH gradient of potency, because the epitope for MAb 16 is at residues 1-16, that for MAb 18 at residues 98-105, and the epitopes of the ineffective group II and III MAbs are within sequence 87-243. Although both HDL subclasses showed that the most potent inhibitory MAbs were those of group I and, among these, that the weakest was MAb 18 (0-14% inhibition), there were a number of differences compared to isolated apoA-I.

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T4-BINDING SITE OF APOA-I

550

Thus, MAb 14 was less potent than MAb 4, and in one case it gave no inhibition, MAb 16 was the strongest inhibitor in only two of the six preparations, and in two of the other preparations, MAb 9 (group III) produced a moderate inhibition (18-27%). Group II MAbs always failed to inhibit (0-3%). Experiments with heparin The effect of heparin, at concentrations up to 5 times the therapeutic serum level, on T4 binding is presented in Table 1. In the case of isolated apoA-I, both equilibrium and photoaffinity labeling showed a maximal inhibition of 8%. These results are consistent with the findings obtained with MAbs, because the sequence 1-76 contains only 8 basic amino acids (or 10 per 100 residues) and is relatively distant from the regions where the basic amino acid content is highest (see Fig. 1). In the case of HDL2 and HDL3, however, heparin resulted in up to 27% inhibition of T4 binding to HDL (equilibrium labeling) or to the 28.3-kDa band (photoaffinity labeling). Quenching of Trp fluorescence by L-T4

Unlabeled L-T4 (10 /xM) quenched the Trp fluorescence of isolated and HDL2-associated apoA-I by 31% and 39%, respectively (data not shown). It should be noted that apoA-I and apoA-II make up about 80% of the protein mass of HDL, and that apoA-II has no Trp residues. Discussion Several lines of evidence indicate that the T4-binding domain of apoA-I is located in the N-terminal third of the molecule. In our studies with isolated apoA-I, only MAbs with epitopes between residues 1-105, particularly

Endo • 1991 Voll28«Nol

MAbs with epitopes between residues 1-86, are effective inhibitors of photoaffinity labeling with T4. The region spanning the first 76 residues has the lowest concentration of basic amino acids, probably accounting for the failure of heparin to prevent T4 binding or to inhibit photoaffinity labeling. The ability of T4 binding to quench tryptophan fluorescence is consistent with this location of the T4-binding site, since three of the four tryptophan residues are in this domain, and the fourth is at residue 108. Since the T4-binding site of transthyretin (2) and probably that of TBG (3-5) are in regions having /3-sheet structure, it is of interest to examine this question in apoA-I. As shown by its computer-predicted secondary structure (Fig. 4), there is very little /3-structure in apoAI, but the highest /3 potential is in the region of residue 18. This overlaps the epitope of MAb 16 (residues 1-16), the most effective inhibitor of photoaffinity labeling by T4. Although most apolipoproteins have little /3-potential, an exception is apoB-100, which has 20-25% /3-sheet structure (15-17) and three T4-binding sites (14). The lipid-binding domain of apoA-I consists of an amphipathic a-helical structure located in the C-terminal two thirds of the molecule (18), and thus, incorporation of apoA-I in the HDL particle would not be incompatible with T4 binding, as we have previously reported (1). The present experiments, however, show differences in MAb inhibition of photoaffinity labeling of apoA-I in HDL compared to isolated apoA-I. Although the group I MAbs are still the most effective inhibitors, the relative effectiveness of the individual MAbs is different and varies in different HDL preparations. Furthermore, MAb 9, which binds to the C-terminal third of apoA-I and has no effect on photoaffinity labeling of isolated apoA-I, does significantly inhibit T4 labeling of

TABLE 1. Effect of heparin on the binding of [125I]T4 to isolated apoA-I, or to three preparations of HDL2 and HDL3 Photoaffinity labeling6'c

Equilibrium labeling0'" Exp 1 None ApoA-I HDL2 1 2 3

Exp 2

Exp 3

None

1 fiM

29,042

26,630 (-8.3)

510

484 (-5.1)

475 (-6.9)

473

441 (-6.8)

41,481 40,576 NDd

31,415 (-24.3) 31,334 (-22.8) ND

ND ND

ND ND 753 (+1.0)

ND ND 608 (-18.0)

ND ND

ND ND 465 (-27.0)

46,166 42,093 ND

38,722 (-16.1) 37,152 (-11.7) ND

ND

ND ND 248 (-4.2)

ND ND 214 (-17.4)

ND ND

10

MM

742

10 jtM

None

637

10

MM

HDL3 1 2 3

ND 259

562

ND ND 490 (-12.8)

0

Net counts per minute in the void volume after Sephadex G-25 chromatography. Percent change is in parentheses. c Densitometry area units of the apoA-I band after autoradiography of SDS-PAGE. d Not done. 6

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T4-BINDING SITE OF APOA-I

551

BETA SHEET POTENTIAL (Chou in< rain APOH F0PROTE1N A-I HUMAN PRECURSOR Window 6

FIG. 4. Computer-predicted /?-sheet potential of human apoA-I. Note the correlation between the peak of (3 structure potential (around residue 18) and the epitope for MAb 16 (residues 1-16).

I 1

I 26

some HDL preparations. It is known that apoA-I undergoes significant conformational changes, such as an increase in a-helical content, upon interaction with lipids (19), and we have previously found (1) that lipids decrease T4 binding to isolated apoA-I. Presumably, the conformational changes alter the proximity of MAb epitopes other than those of group II to the T4-binding site and account for the effects that we have observed. They also could account for the observation that heparin inhibits T4 binding to apoA-I only when it is lipid associated. An altered relation between the C-terminal region of apoA-I and the T4binding site could account for both the inhibitory effect of MAb 9 and the heparin effect (see the cluster of basic residues in the region between amino acids 164-208 in Fig. 1). Other workers have described different effects of the same MAb on the isolated and lipid-associated forms of another apolipoprotein, apoE (9). Recent evidence demonstrates the existence in apoE of two structural domains separated by a region highly susceptible to proteolytic cleavage: a stable N-terminal domain (residues 20-165), whose last approximately 20 residues mediate the interaction of apoE with the apoB/E receptors, and a less stable C-terminal domain (residues 225-299) which contains amphipathic a-helices and, thus, constitutes the major lipid-binding portion of apoE (20, 21). The /3-sheet structure content of apoE is about 11% and, as shown in Figs. 7 and 1 of Refs. 20 and 21, respectively, is contained in the N-terminal portion. In view of the homology among apolipoproteins, including apoA-I and apoE (18), and since we have preliminarily demonstrated that apoE is another thyroid hormone-binding protein (22), it may be suggested that the T4-binding region of both proteins is in the N-terminal domains, distinct from C-terminal lipid-binding domains. We are currently investigating the T4-binding site of apoE. The variability in the MAb pattern of inhibition of

76

159

288

1

I

126

176

250

I

I

226 243

Mature Peptide

photoaffinity labeling even within different preparations belonging to the same HDL subclass may be related to the known polydisperse nature of the lipoproteins, in particular those belonging to the HDL class (23). Our findings also can be related to the unequal distribution of T4 within the HDL class in human plasma (10) and might have implications for the physiological role of T4lipoprotein interactions. Acknowledgments We would like to thank Dr. L. Curtiss (La Jolla, CA) for her generous gift of the monoclonal antibodies, Dr. D. Sackett (NIDDK, NIH) for the tryptophan quenching experiment, Dr. A. Facchiano (NICHHD, NIH) for the computer-aided prediction of the /3-sheet potential of apoA-I, and Dr. B. Brewer, Jr. (NHLBI, NIH), for having read the manuscript before submission.

References 1. Benvenga S, Cahnmann HJ, Gregg RE, Robbins J 1989 Characterization of the binding of thyroxine to high density lipoproteins and apolipoprotein A-I. J Clin Endocrinol Metab 68:1067-1072 2. Blake CCF, Oatley SJ 1978 Protein-DNA and protein-hormone interactions in prealbumin: a model of the thyroid hormone nuclear receptor. Nature 268:115-120 3. Tabachnick M, Perret V 1981 Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide isolated from a proteolytic digest of the derivatized protein. Biochem Int 15:409-417 4. Flink IL, Bailey TJ, Gustafson TA, Markham BE, Morkin E 1986 Complete amino acid sequence of human thyroxine-binding globulin deduced from cloned DNA: close homology to the serine antiprotease. Proc Natl Acad Sci USA 83:7708-7712 5. Loeberman H, Tokuoka R, Deisenhofer J, Huber R 1984 Human al-proteinase inhibitor. Crystal structure analysis of two crystal modifications: molecular model and preliminary analysis of the implications for functions. J Mol Biol 177:531-556 6. Krul ES, Schonfeld G 1986 Immunochemical methods for studying lipoprotein structure. In: Segrest JP, Albers JJ (eds) Methods in Enzymology. Academic Press, Orlando, FL, 1990 vol 128:527-552 7. Benvenga S, Cahnmann HJ, Robbins J 1990 Localization of the thyroxine binding sites in apolipoprotein B-100 of human lowdensity lipoproteins. Endocrinology 127:2024-2046 8. Wenzel K 1981 Pharmacological interference with in vitro tests of thyroid function. Metabolism 30:717-732

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9. Weisgraber KH, Rail Jr SC, Mahler RW, Milne RW, Marcel YL, Sparrow JT 1986 Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3. J Biol Chem 261:20682076 10. Benvenga S, Gregg RE, Robbins J 1988 Binding of thyroid hormones to human plasma lipoproteins. J Clin Endocrinol Metab 67:6-16 11. Curtiss LK, Edington TS 1985 Immunochemical heterogeneity of human plasma high density lipoproteins: identification with apolipoprotein A-I and A-II specific monoclonal antibodies. J Biol Chem 260:2982-2993 12. Curtiss LK, Smith RS 1987 Immunochemical Heterogeneity of HDL. Proceedings of the Workshop on Lipoprotein Heterogeneity. NIH Publication 87-2646:363-377 13. Curtiss LK, Smith RS 1988 Localization of two epitopes of apolipoprotein A-I that are exposed on human high density lipoproteins using monoclonal antibodies and synthetic peptides. J Biol Chem 263:13779-13785 14. Benvenga S, Cahnmann HJ, Gregg RE, Robbins J 1989 Binding of thyroxine to human plasma low density lipoproteins through specific interaction with human apolipoprotein B (apoB-100). Biochimie 71:263-268 15. Knott TJ, Pease RJ, Powell LM, Wallis SC, Rail Jr SC, Innerarity TL, Blackhart B, Taylor WH, Marcel Y, Milne R, Johnson D, Fuller M, Lusis AJ, McCarthy BJ, Mahley RW, Levy-Wilson B, Scott J 1986 Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature 323:734-

738 16. Yang C-Y, Chen S-H, Gianturco SH, Bradley WA, Sparrow JT, Tanimura M, Li W-H, Sparrow DA, DeLoof H, Rosseneu M, Lee F-S, Gu Z-W, Gotto Jr AM, Chan L 1986 Sequence, structure, receptor binding, domains and internal repeats of human apolipoprotein B-100. Nature 323:738-742 17. Law SW, Grant SM, Higuchi K, Hospattanakar A, Lockner K, Lee N, Brewer Jr HB 1986 Human liver apolipoprotein B-100 cDNA. Complete nucleic acid and derived amino acid sequence. Proc Natl Acad Sci USA 83:8142-8146 18. Breslow JL 1987 Lipoprotein genetics and molecular biology. In: Gotto Jr AM (ed) Plasma Lipoprotein. Elsevier, Amsterdam, pp 359-397 19. Laggner P 1981 Physicochemical characterization of high density lipoproteins. In: Day CE (ed) High Density Lipoproteins. Marcel Dekker, New York, pp 43-72 20. Wetterau JR, Aggerbeck LP, Rail Jr SC, Weisgraber KH 1988 Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains. J Biol Chem 263:6240-6248 21. Mahley RW 1988 Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240:622-630 22. Benvenga S, Robbins J 1990 Enhancement of thyroxine entry into low density lipoprotein (LDL) receptor-competent fibroblasts by LDL: an additional mode of entry of thyroxine into cells. Endocrinology 126:933-941 23. Patsch JR, Gotto Jr AM 1987 Metabolism of high density lipoproteins. In: Gotto Jr AM (ed) Plasma Lipoproteins. Elsevier, Amsterdam, pp 221-259

Endocrine Society Members In Remembrance John P. Allen, M.D. Leela S. Craig, M.D., F.A.C.P. Larry L. Ewing, Ph.D. Norbert Freinkel, M.D. Masanobu Honda, M.D. Jerrold D. Hydovitz, M.D. Anthony J. Izzo, M.D. Richard Scott Jaeckle, M.D. Joseph P. Kriss, M.D.

Endo • 1991 Voll28«Nol

Grant W. Liddle, M.D.* Juan Carlos Penhos, M.D. Rosaline V. Pitt-Rivers, Ph.D. Ora Mendelsohn Rosen, M.D. Diane H. Russell, Ph.D. Sheila F. Stewart, Ph.D. Diana E. Van Orden, M.D. Frank G. Young, D.Sc. * Past President

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