THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1987 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 262. No. 35, Issue of December 15, pp. 16838-16847 1987 Printed in d.S.A.

Characterization of the Chicken Oocyte Receptor for Low and Very Low Density Lipoproteins*

(Received for publication, June 8, 1987)

Rajan George, Dwayne L. Barber$, and Wolfgang J. Schneiders From the Department of Biochemistry, The University of Alberta, Edmonton, Alberta, Canada T6G 2H7

The chicken oocyte receptor for low and very low density lipoproteins has been identified and character- ized. Receptor activity present in octyl-j3-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent M, of 96,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high af- finity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, sura- min, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Fur- thermore, a structural relationship between the mam- malian and avian receptors was revealed by immuno- blotting: polyclonal antibodies directed against the pu- rified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.

In oviparous (egg-laying) species, the developing embryo is absolutely dependent on the egg components for its nutri- tional requirements. Yolk, the complex storage component of the oocyte, serves as the major food source. Most, if not all, yolk proteins are synthesized in and secreted from the liver, transported as macromolecules in the plasma to the oocyte, where specific surface receptors are thought to mediate their cellular uptake (1-3). Evidently, beyond a normal genetic background, there are two additional requirements for normal growth of the embryo. First, the composition ( i e . the relative amounts) of yolk proteins must satisfy the complex needs of the rapidly growing organism; second, the absolute amount of yolk available is to demarcate the endpoint of embryonic growth. At birth, egg components have been completely con- verted into a living animal.

Our efforts are directed toward gaining a detailed knowledge of the mechanisms underlying the control of oocyte growth. At least two possible modes of regulation can be conceived first, at the level of hepatic yolk protein synthesis, which is

* This research was supported by grants from the Alberta Heritage Foundation for Medical Research and Grant MA-9083 from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Recipient of a Studentship Award from the Alberta Heritage Foundation for Medical Research.

3 Scholar of the Alberta Heritage Foundation for Medical Re- search. To whom correspondence should be addressed Dept. of Bio- chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

under the control of estrogens (reviewed in Refs. 4, 5). The other site of oocyte growth regulation may be at the level of expression of proteins mediating the transport of the yolk components into the oocyte. Existing evidence strongly sug- gests that at least the major yolk precursors, namely vitello- genin, a lipophosphoglycoprotein (6-8), and triglyceride-rich lipoproteins of the very low density class (9-13), are delivered into the oocyte by the process of receptor-mediated endocy- tosis (14). In birds, additional components isolated from yolk such as transferrin (15,16), riboflavin carrier protein (17-19), retinol-binding protein (20), thiamin-binding protein (21,22), and certain biotin-binding protein(s) (23) share structural and immunological properties with corresponding plasma pro- teins, and are presumably also taken up into the oocyte by processes involving specific receptors. The presence of oocyte plasma membrane receptors for these plasma components, as well as for immunoglobulin G, has often been implied and preliminary evidence for the existence of such receptors pro- vided (6, 10,24-26), but a thorough biochemical characteriza- tion of any of these important molecules is lacking.

We have chosen the chicken (Gallus domesticus) for studies on the regulation of receptor-mediated endocytosis and its role in oogenesis for two groups of reasons. First, from an experimental point of view, because (i) hepatically synthesized yolk proteins, in particular very low density lipoprotein (VLDL)’ and vitellogenin, can easily be purified either from the plasma of laying hens or estrogen-treated roosters; (ii) many of the less abundant proteins (e.g vitamin-binding proteins) have been purified and characterized from chicken plasma or yolk; and (iii) chicken oocytes have an enormous growth rate: 1.5-2 g of protein are taken up per day for the last 7 days before oviposition (2, 27). These giant cells, thus, can be expected to contain substantial amounts of proteins functioning in endocytosis, facilitating their biochemical characterization. Second, the chicken system is of interest from a physiological point of view. Estrogens play a dual role: on one hand, they stimulate hepatic output of lipid-rich particles (VLDL and vitellogenin) which, if not removed from the bloodstream, have detrimental effects as evidenced by the rapid formation of aortic plaques in estrogen-treated roosters (28) and in chickens with hereditary hyperlipidemia (29); on the other hand, the same lipoproteins are essential for the reproductive effort of the female. There are additional dis- tinctive features of avian lipoprotein metabolism worthy of investigation. For example high density lipoproteins, which carry about 90% of all plasma lipids in young birds (301, almost disappear upon maturation of hens when VLDL be- comes the prevalent lipid carrying plasma protein. Thus, the role of triglyceride-rich lipoproteins in avian metabolism in

The abbreviations used are: (V)LDL, (very) low density lipopro- tein; HDL, high density lipoprotein; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride.

16838

Lipoprotein Receptors in Oocytes 16839

general, and in oogenesis in particular, is of considerable interest.

Here, we report on the characterization of the chicken oocyte membrane receptor for LDL and VLDL. We used detergents to solubilize the receptor and the technique of ligand blotting (31) to identify the protein.

EXPERIMENTAL PROCEDURES

Materiuls-We obtained DEAE-cellulose DE52 from Whatman; Sephadex G-25 columns PD-10 from Pharmacia Biotechnologies, Inc.; egg phosphatidylcholine (Cat. No. P-5388), octyl-p-D-glucoside, PMSF, leupeptin, Triton X-100, 17a-ethinylestradiol, and bovine serum albumin (Cat. No. A-7030) from Sigma; M, standards from Bethesda Research Laboratories and Sigma; nitrocellulose paper BA 85 from Schleicher & Schuell Pronase (Cat. No. 165921) from Boeh- ringer Mannheim; Nuflow cellulose acetate membrane filters N25/45 from Oxoid Ltd., Basingstoke, England; sodium ["']iodide (11-17 mCi/pg) from Edmonton Radiopharmaceutical Centre, Edmonton, Alberta, Canada; and suramin from FBA Pharmaceuticals, New York; LDL receptor from bovine adrenal cortex was purified as described (32); other materials were obtained from previously reported sources (31).

Animals and Diets-White Leghorn layers (8-18 months old) were obtained from a local poultry farm and maintained on layer mash, with a light period of 12 h. White Leghorn roosters (4-8 weeks old) were kindly provided by Dr. V. E. Barracos, Department of Animal Sciences, The University of Alberta, and maintained on grower mash, with a light period of 12 h. For raising antibodies, we used adult female New Zealand White rabbits.

Lipoproteins-Human VLDL ( p < 1.006) and LDL (1.019 < p < 1.063 g/ml) were prepared from plasma by differential ultracentrifu- gation (33). For the preparation of chicken LDL and VLDL, we administered 17a-ethinylestradiol dissolved in propyleneglycol (10 mg/kg body weight) to 5-8-week-old roosters by intramuscular injec- tion; 72 h later the birds were killed by decapitation and blood was collected in 10 mM EDTA, 1 mM PMSF, and 5 p M leupeptin. Alter- natively, we obtained blood from laying hens. Plasma was prepared and subjected to ultracentrifugation for 36 h at 4 "C and 200,000 X g.". The floating lipoprotein fraction was mixed with 0.15 M NaCl, 0.2 mM EDTA, 1 mM PMSF, and 5 p~ leupeptin ( p = 1.006 g/ml), and a second centrifugation step at 200,000 X g., for 24 h performed. Chicken VLDL was recovered from the top of the centrifuge tube. Chicken LDL (1.025 < p < 1.063) was prepared from the infranate of the initial centrifugation step by consecutive centrifugation at den- sities 1.063, 1.025, and 1.063, respectively. For the preparation of chicken HDL (1.15 < p < 1.21), plasma was obtained from 5-8-week- old untreated roosters and HDL prepared by consecutive ultracen- trifugation steps at densities 1.006 (36 h), 1.21 (24 h), 1.15 (24 h), and 1.21 (36 h), respectively, for the indicated times at 4 'C and 200,000 X g., each. Reductively methylated lipoproteins were prepared by treatment with formaldehyde plus sodium borohydride (34). Lip- oprotein concentrations are expressed in terms of protein content. '%I-labeled lipoproteins were prepared by the iodine monochloride method as previously described (35). All lipoproteins were dialyzed exhaustively against buffer containing 0.15 M NaCl, 0.2 mM EDTA, pH 7.4, before use and stored at 4 "C. Apoprotein VLDL-I1 was purified by detergent-extraction of chicken VLDL as described else- where.'

Preparation of Oocyte Membranes-All operations were performed at 0-4 "C. Follicles (3-15-mm in diameter) were rapidly transferred to buffer containing 20 mM Tris-HC1 (pH 8), 1 mM CaCl', 150 mM NaCl, 1 mM PMSF, and 2 p~ leupeptin (buffer A). Theca externa and theca interna were removed by dissection and the yolk extruded through an incision. The remaining material, consisting of basal lamina, granulosa cell layer, perivitelline layer, and oocyte plasma membrane, was rinsed with buffer A until the wash fluid was free of yolk. The membranes were minced with scissors, and subjected to homogenization in 10 ml of buffer A per g wet weight with a Polytron homogenizer (probe P-10) for 30 s at setting 5, followed by two periods of 20 s each at setting 8. Large debris were removed by centrifugation at 5,000 X g for 5 min and the resulting supernatant subjected to centrifugation at 100,000 X g for 1 h. The membrane pellets were suspended in buffer A by aspiration through a 22-gauge

' R. George, J. Nimpf, and W. J. Schneider, manuscript in prepa- ration.

needle and resedimented by centrifugation at 100,000 X g for 1 h; this was repeated once. The washed membrane pellet was quickly frozen in liquid Nz and stored at -70 "C for up to 3 months before use.

Solubilization of Chicken Oocyte Membrane Proteins-All opera- tions were carried out at 0-4 "C. In a typical solubilization experi- ment, membrane pellets obtained from the ovary of one chicken (weight of follicles, 7-10 g) were suspended in 2 ml of buffer contain- ing 250 mM Tris-maleate (pH 6), 2 mM CaCL, 1 mM PMSF, and 2 p~ leupeptin by aspiration through a 22-gauge needle. The suspension was sonicated twice for 20 s (Sonifier model W 185, Heat-System- Ultrasonics, Inc.) with a microprobe at setting 6. Reagents were then added to adjust the suspension to a final volume of 4 ml containing the following components: 7-10 mg of protein/ml, 125 mM Tris- maleate (pH 6), 2 mM CaCl,, 0.16 M NaCl, 0.5 mM PMSF, 1 p M leupeptin, and 36 mM octyl-@-D-glucoside. The suspension was stirred at 4 "C for 10 min, and undissolved material was removed by centrif- ugation at 100,000 x g for 60 min. The clear supernatant, designated octyl glucoside extract, was either quickly frozen in liquid NZ and stored at -70 "C for up to 2 months or used immediately for ligand binding experiments as described below.

Antibodies-Polyclonal rabbit anti-LDL receptor antibodies were raised by injection of 20 pg of purified bovine LDL receptor (32) dissolved in 300 ~10.15 M NaCI, and emulsified with 300 pl of Freund's complete adjuvant on day 0, followed by immunizations at days 14, 28, and 35 with 10 pg of receptor protein each in Freund's incomplete adjuvant. IgG was prepared by chromatography on Protein A-Seph- arose (37) from serum obtained on days 42 and once every 2 weeks thereafter. No loss in titer was observed over a period of 4 months. Nonimmune IgG was obtained from the same animal before immu- nization. Goat-anti rabbit IgG (Cooper No. 0612-3151) was radiola- beled with lZ5I by the iodogen method as previously described (37).

Electrophoresis, Transfer to Nitrocellulose and Ligand Blotting- One-dimensional SDS gel electrophoresis according to Laemmli (38) was conducted on slab gels (16 X 12 X 0.15 cm) with the concentra- tions of polyacrylamide as indicated in the figure legends. For ligand blotting experiments, the samples did not contain reducing agents and were not heated prior to application unless stated otherwise. In other experiments, samples contained 10 mM dithiothreitol and were heated to 90 "C for 5 min. Electrophoresis was performed at 25-35 mA/slab at 10 "C for 4-5 h. Gels were calibrated with all or some of the following M, standards as indicated in the figures: myosin, 200 kDa; @-galactosidase, 116 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; @- lactoglobulin, 18 kDa; and lysozyme, 14 kDa. Electrophoretic transfer of proteins to nitrocellulose was performed and protein bands on nitrocellulose were stained with Amido Black 10 B as described previously (39). Ligand blotting was carried out with 5% (w/v) bovine serum albumin as blocking agent as described previously (31), except that 'T-labeled chicken or human lipoproteins were used as ligands. The concentrations and specific radioactivities of the ligand used in the incubation mixtures are indicated in the figure legends. Autora- diographs were obtained by exposing the dried nitrocellulose paper to Kodak XAR-5 film at room temperature for the indicated times.

Filter Assay for Lipoprotein Binding to Oocyte Extracts-The solid- phase filtration assay described previously (40) for the binding of lZ5I- LDL to bovine adrenocortical extracts was adopted. Briefly, lipopro- tein-binding sites present in an aliquot of the oocyte membrane octyl glucoside extract were precipitated by adjusting the octyl glucoside concentration to 4.5 mM by addition of 7 volumes of buffer containing 50 mM Tris-maleate (pH 6), and 2 mM CaC12; the precipitate was collected by centrifugation at 100,000 X g for 60 min at 4 "C. The precipitate was resuspended by aspiration through a 22-gauge needle in buffer containing 20 mM Tris-HC1 (pH 8), 50 mM NaCl, 1 mM CaCI, and used in the filter assay. If not indicated otherwise, the standard assay mixture (pH 8) contained in a volume of 100 p l 12.5 mM Tris-HC1, 25 mM NaCl, 2 mM CaCl', 16 mg/ml bovine serum albumin, the indicated amounts of protein of precipitated octyl glu- coside extract, and the indicated concentrations of 1'61-labeled lipo- protein. Incubation was for 2 h at 24 'C, and free ligand was separated from receptor bound by filtration as described (40).

Other Assays-The protein content of samples containing no octyl glucoside was determined by the method of Lowry et al. (41). The protein contents of samples containing octyl glucoside were measured by modifications of the Lowry procedure as previously described (42) with bovine serum albumin as standard. Triglyceride (43), total cholesterol (44), and phospholipid (45) in isolated lipoprotein frac- tions was determined as described in the indicated references.

16840 Lipoprotein Receptors in Oocytes

RESULTS

Isolation and Characterization of Chicken Lipoproteins- Fig. 1 shows the results of analysis by SDS-polyacrylamide gel electrophoresis of the apoprotein contents of chicken HDL, LDL, and VLDL isolated as described under “Experi- mental Procedures.” Apolipoprotein A-I (molecular mass, 28 kDa) was the sole detectable protein component of high density lipoprotein (1.15 < p < 1.21) isolated from untreated roosters (Fig. 1, lane A ) or immature hens (not shown). A total of four ultracentrifugal separation steps was required to obtain a pure high density lipoprotein fraction, characterized by the absence of apolipoproteins other than apolipoprotein A-I. LDL, isolated at densities 1.025-1.063 as described under “Experimental Procedures” did not contain detectable amounts of apolipoprotein VLDL-I1 (lane C). When VLDL from estrogen-treated roosters (Fig. 1, lane D) was prepared by two sequential ultracentrifugal flotation steps a t p = 1.006, the lipoprotein particles contained only high molecular weight apolipoprotein B and apolipoprotein VLDL-I1 as protein com-

Mr -1 0 -3

- 200

- 97

- 68

- 43

-29

-18 -14

A B C D E FIG. 1. SDS-polyacrylamide gel electrophoresis of chicken

lipoproteins. Chicken lipoprotein fractions were purified as de- scribed under “Experimental Procedures,” and aliquots delipidated by extraction with 20 volumes of ice-cold chloroform/methanol (2:1, v/v) prior to electrophoresis on a 5-20% SDS-polyacrylamide gradient gel. Samples contained 10 mM dithiothreitol and were heated to 90 “C for 5 min. Lune A, 6 pg of protein of rooster HDL; lane C, 10 pg of protein of laying hen LDL; lane D, 18 pg of protein of VLDL from estrogen-treated rooster; lane E, 8 pg of apolipoprotein VLDL-11; and lane B, M, standards are indicated. The proteins were stained with Coomassie Blue.

ponents. Apolipoprotein VLDL-I1 is a disulfide-linked hom- odimer of identical 82-residue polypeptides (46); the disulfide linkage is highly resistant to reduction (30). In our gradient gel system (4.5-1896 polyacrylamide), the dimer had a mobil- ity correlating to an M , of 16,000 with the monomer having an apparent M, of 9,500, as confirmed by analysis of isolated apolipoprotein VLDL-I1 (lane E). With our isolation proce- dure, there was no apparent difference between the apoprotein patterns of LDL and VLDL isolated from laying hens and estrogen-treated roosters. Furthermore, we did not detect measurable differences in binding characteristics between lipoproteins from laying hens or estrogen-treated roosters (see below); thus, LDL and VLDL from both sources were used in subsequent experiments.

Binding of ‘251-Labekd Chicken Lipoproteins to Oocyte Membrane Extracts-When octyl glucoside extracts of oocyte

‘esI -ER LDL oJg/ml)

FIG. 2. Saturation curve for the binding of chicken 12sI- VLDL and 1251-LDL to chicken oocyte membrane octyl glu- coside extracts. Each assay tube contained the standard assay mixture (100 pl ) with 15.4 pg of protein of precipitated octyl glucoside extract and the indicated concentration of radiolabeled ligand in the presence (A) or absence (0) of 20 mM EDTA. In Panel A, the ligand was laying hen Iz5I-VLDL, (specific activity, 82 cpm/ng) and nonspe- cific binding was determined from incubations containing 1.6 mg/ml of unlabeled laying hen VLDL (0); in Panel B, the ligand was estrogen-treated rooster ‘%I-LDL (specific activity, 70 cpm/ng), and nonspecific binding was determined from incubations containing 1.6 mg/ml of unlabeled LDL (0) from the same animal. The amount of receptor-bound radiolabeled ligand was determined as described un- der “Experimental Procedures.” High affinity binding (W) was cal- culated by subtracting nonspecific binding (0) from total binding (0). Each data point represents the average of duplicate determina- tions.

Lipoprotein Receptors in Oocytes 16841

membranes were analyzed for their ability to bind lZ5I-chicken VLDL or lZ5I-chicken LDL by the solid-phase filter assay under standard conditions, we found that both ligands bound with high affinity and in saturable fashion. The presence of either 20 mM EDTA or excess unlabeled ligand obliterated the saturable binding component of both 9 - V L D L (Fig. 2 A ) and '"I-LDL (Fig. 2B), and only nonsaturable linear compo- nents were observed. Analysis of the high affinity binding data according to Scatchard (Table I) revealed that both LDL and VLDL interacted with a single class of binding sites. In addition, lipoproteins from both laying hens and estrogen- treated roosters bound with similar kinetic characteristics. Table I demonstrates that the average concentrations of li- gand protein giving half-maximal binding to the saturable site were similar (10.6 pg/ml for LDL, and 13.5 pg/ml for VLDL), as were the maximum amounts of ligand bound (67

TABLE I Parameters for lipoprotein binding to chicken oocyte

membrane octyl glucoside extracts Saturation curves for the binding of radiolabeled lipoproteins to

precipitated octyl glucoside extracts were obtained as described under "Experimental Procedures" and the legends to Figs. 2 and 6. K d and B , values were calculated from Scatchard plots (36) of high affinity binding data for the indicated lipoproteins. LH, laying hen, ER, estrogen-treated rooster, and H, human. Each value represents the average of the results from two independent sets of binding experi- menta with the same preparation of precipitated octyl glucoside entract.

Lipoprotein K d B ,

Mlml pg f mg protein LH-VLDL 13.0 71 ER-VLDL 14.0 79

LH-LDL 10.1 69 ER-LDL 11.0 64

H-VLDL -200 H-LDL

N.D." 43 18

N.D., not determined.

20 40 60 80 110 1 3 0 150 170

TIME (minutes)

FIG. 3. Binding of "I-VLDL to oocyte receptors. Time courm and effect of suramin. Each assay tube contained the standard assay mixture (100 pl) with 21 pg of protein of precipitated octyl glucoside extract and 5 pg/ml of laying hen '"I-VLDL (200 cpm/ng) in the absence (0) or presence (A) of 1 mg/ml of unlabeled VLDL. After incubation at 24 "C for the indicated times, receptor- bound '"I-VLDL was measured as described under "Experimental Procedures." At 110 min (arrow), one set of tubes (0) received suramin (final concentration, 2 mg/ml), and receptor-bound '%I- VLDL was determined in the presence or absence of (0) of suramin at the indicated timepoints. The inset demonstrates the inhibitory effect of suramin on receptor binding of '%I-VLDL. Suramin was added to assay tubes to give the indicated concentrations before the addition of '"I-VLDL, and receptor-bound '%I-VLDL was determined after incubation at 24 'C for 2 h.

pg/mg of protein and 75 pg/mg of protein for LDL and VLDL, respectively). The number of binding sites and their affinity were similar at 4,24, and 37 "C. The pH optimum for binding was 8.0, and the amount of binding was proportional to the amount of protein in the precipitated octyl glucoside extract up to at least 50 pg/assay tube (data not shown).

Previously, it has been observed that suramin, a polysul- fated polycyclic hydrocarbon, inhibits the binding of human LDL to the LDL receptor and is able to dissociate LDL rapidly from the receptor when added after binding had occurred (47). We, therefore, tested the effects of suramin on the binding of chicken LDL and VLDL to oocyte octyl glu- coside extracts. Fig. 3 demonstrates that the binding of lZ5I- VLDL to oocyte extracts displayed the same characteristics as the interaction of mammalian LDL with its receptor. In the absence of suramin, binding of lZ5I-VLDL to precipitated octyl glucoside extracts at 24 "C reached completion within 40 min, and no measurable dissociation occurred over a period of 4 h. However, upon addition of suramin, the bound lZ5I- VLDL was rapidly released from its receptor. When increas- ing amounts of suramin were added at the beginning of the incubation, complete inhibition of 'T-VLDL binding was observed at a concentration of 1.25 mg/ml (Fig. 3, inset). The same results were obtained with 'T-chicken LDL as ligand (data not shown).

Solubilization and Characterization of the Oocyte Receptor- When detergent extracts from oocyte membranes were pre- pared by the use of octyl-p-D-glucoside, measurable lipopro- tein binding activity could be recovered in a precipitate ob- tained by decreasing the detergent concentration to below its critical micellar concentration, as described under "Experi- mental Procedures." Soluble extracts retained full T-VLDL and -LDL binding activity for at least 2 months when stored at -70 "C. Heating of extracts to 90 "C for 5 min destroyed the receptor activity. Furthermore, the activity was destroyed by incubation of the precipitated extract with Pronase in a concentration-dependent fashion (Fig. 4). Incubation for 10 min at 37 "C with 25 pg/ml of Pronase completely abolished

"I IO n I

d 0 5 IO 15 20 25 30 3:

[PWASE] , )rg 1 ml

FIG. 4. Effect of Pronase treatment of precipitated oocyte membrane octyl glucoside extracts on Iz6I-VLDL binding. Aliquots (22 pg of protein) of precipitated oocyte membrane octyl glucoside extract were suspended in Dulbecco's phosphate-buffered saline plus 1 mM CaC12 and incubated with the indicated concentra- tions of Pronase for 10 min at 37 "C in a volume of 40 pl. The action of Pronase was stopped by addition of 40 pl of ice-cold buffer con- taining 12.5 mM Tris-HC1, (pH 8.0) 25 mM NaCl, 2 mM CaC12, and 80 mg/ml bovine serum albumin. Each tube received 5 pg/ml of "'1- VLDL (356 cpm/ng) in the absence (0) or presence (A) of 1 mg/ml of unlabeled VLDL. After incubation at 4 "C for 2 h, receptor-bound '%I-VLDL was measured as described under "Experimental Proce- dures."

16842 Lipoprotein Receptors in Oocytes

A B C D E F G

-200

T -97 0

7

FIG. 5. Ligand blotting of oocyte membrane octyl glucoside extracts. Oocyte membrane octyl glucoside extract (17 pg of protein/ lane) was subjected to electrophoresis in a 4.5-18% SDS-polyacryl- amide gradient gel under nonreducing conditions, followed by transfer to nitrocellulose. Ligand blotting was performed as described under "Experimental Procedures." The strips were incubated in buffer with 5 pg/ml of estrogen-treated rooster IZ5I-VLDL (180 cpm/ng) and the following additions: A, none; E , 0.5 mg/ml unlabeled chicken VLDL; C, 0.5 mg/ml unlabeled chicken LDL; D, 0.5 mg/ml unlabeled human LDL; E, 0.5 mg/ml unlabeled human methyl-LDL; and F, 0.5 mg/ml unlabeled chicken HDL; and G, 5 mg/ml suramin. Exposure to XAR- 5 film was for 24 h at room temperature. M. standards are indicated. After obtaining this and short-time exposed autoradiographs (see text), the radioactive bands were cut out from the nitrocellulose strips and their radioactivity determined. The radioactivity in an equally sized piece representing the background was determined for each strip and subtracted. The resulting radioactivities were: 15,620, 970, 930, 13,210, 14,990, 14,870, and 30 cpm for lanes A-G, respectively.

'**I-WMAN LDL (w / ml)

FIG. 6. Saturation curve for the binding of human "I-LDL to chicken oocyte membrane octyl glucoside extracts. Each assay tube contained the standard assay mixture (100 pl) with 24 pg of protein of precipitated octyl glucoside extract, and the specified concentrations of human '=I-LDL (specific activity, 235 cpm/ng) in the presence (A) or absence (0) of 20 mM EDTA or in the presence of 2.25 mg/ml of unlabeled human LDL (0). Receptor-bound '=I- LDL was determined as described under "Experimental Procedures." High affinity binding (U) was calculated by subtracting nonspecific binding (0) from to ta l binding (0). Each data point represents the average of duplicate determinations.

1 2 3

Chicken- I LDL VLDL

4 5 6 7 a

.200

,116

,97

68

43

FIG. 7. Ligand blotting of bovine and chicken lipoprotein receptors. Purified bovine receptor (0.8 pg/lane; lanes I, 3,5, and 7) and chicken oocyte membrane octyl glucoside extract (35 pg protein/ lane; lanes 2, 4, 6 and 8) were subjected to electrophoresis on 4.5- 18% SDS-polyacrylamide gradient gels under nonreducing condi- tions, followed by transfer to nitrocellulose, and ligand blotting as described under "Experimental Procedures." The strips were incu- bated in buffer containing the following radiolabeled lipoproteins: lanes I and 2, chicken LDL (15 pg/ml; specific activity, 90 cpm/ng); lanes 3 and 4, chicken VLDL (10 pg/ml; specific activity, 115 cpm/ ng) (both chicken lipoproteins were prepared from plasma of estro- gen-treated roosters); lanes 5 and 6, human VLDL (15 pglml; specific activity, 180 cpm/ng); and lanes 7 and 8, human LDL (15 pg/ml; specific activity, 250 cpm/ng). Exposure to XAR-5 film was for 18 h at room temperature. M, standards are indicated.

specific VLDL binding activity and lowered the small amount of nonspecific binding observed in the presence of excess unlabeled ligand. These findings suggested that the oocyte activity for the binding of LDL and VLDL resided on a membrane protein.

In order to identify the receptor protein, we applied the technique of ligand blotting (31). Oocyte membrane proteins were separated by SDS-polyacrylamide gel electrophoresis, the proteins electrophoretically transferred to nitrocellulose, and the nitrocellulose replicas incubated with radiolabeled ligand in the presence or absence of other components fol- lowed by autoradiography, as described under "Experimental Procedures." When aliquots of octyl glucoside extracts from oocyte membranes were analyzed by this technique with '"1- VLDL as ligand, a protein with a M, of about 95,000 was shown to bind the radiolabeled lipoprotein (Fig. 5, lane A) . Binding of VLDL was markedly reduced when excess unla- beled chicken VLDL ( l a n e B ) or chicken LDL ( l a n e C) were present in the incubation medium. As shown by quantification of receptor-bound '"I-VLDL radioactivity (see legend to Fig. 5), human LDL showed only a small effect on the binding of '"I-chicken-VLDL when added to the incubation medium a t the same concentration as the chicken lipoproteins (Fig. 5, lane D). While human LDL inhibited by 15%, methylated human LDL ( l a n e E ) had almost no effect on '"1-VLDL binding to the 95-kDa protein. Chicken HDL also did not significantly reduce the amount of receptor-bound '"1-VLDL ( l a n e F). Confirming the binding data of Fig. 3, suramin at a

Lipoprotein Receptors in Oocytes 16843

I ER VLDL 0 0.25 0.5 0.75 1.0

UNLABELED LIPOPROTEINS ( mg/ml 1

I I 0 0.25 0.5 0.75 1.0

UNLABELED LIPOPROTEINS ( mp/ ml 1

0 0.02 0. I 0.2

UNLABELED LIPOPROTEINS (mg/ml)

FIG. 8. Competitive binding of chicken and human lipopro- teins to chicken oocyte receptors. Each assay tube contained the standard assay mixture (100 pl) with 16 pg of protein of precipitated octyl glucoside extract and the following radiolabeled ligands: Pawl A, '=I-VLDL from estrogen-treated rooster (8 pg/ml; specific activity, 115 cpm/ng) in the presence or absence of the indicated concentra- tions of unlabeled human LDL (O), rooster HDL (0), and LDL (A) or VLDL (A) from estrogen-treated rooster; Panel B, '''I-LDL from estrogen-treated rooster (14 pg/ml; specific activity, 90 cpm/ng) in the presence or absence of the indicated concentrations of unlabeled

concentration of 5 mg/ml strongly inhibited the binding of lZ5I-VLDL to the 95-kDa protein (Fig. 5, lane G). It should be noted that the autoradiograph in Fig. 5 is the result of pur- posely overexposing the film (exposure time, 24 h). Autora- diography for shorter times revealed somewhat sharper bands in lanes A, D, E, and F, but failed to demonstrate any bands in lanes B and C. The ligand-blotting data together with the results of Fig. 2 suggested that chicken VLDL and LDL both bind to the 95-kDa membrane protein with high affinity. Furthermore, the results of these experiments raised the possibility that the same protein may also recognize the human LDL, albeit with much lower affinity than chicken lipoproteins. This possibility was tested in two experiments as described below.

Binding of lZ5I-Labeled Human Lipoproteins to Oocyte Mem- brane Extracts-First, the binding characteristics of human lZ5I-LDL to precipitated oocyte membrane octyl glucoside extracts were determined by the solid-phase filtration assay (Fig. 6). We found that binding of human 'T-LDL to oocyte membrane extracts was saturable. However, compared with the binding of '251-labeled chicken lipoproteins, the binding of human '251-LDL was of considerably lower affinity and showed a maximum amount of binding that was only about 25% that of the chicken lipoproteins. For comparison, binding constants for all lipoproteins tested in binding assays are summarized in Table I.

Second, we tested whether the 95-kDa oocyte membrane protein that bound chicken LDL and VLDL was also respon- sible for the binding of human lipoproteins of the low and very low density class. To this end, we used ligand blotting to compare the binding of lZ5I-labeled chicken LDL and VLDL, as well as human LDL and VLDL to the purified bovine LDL receptor with their binding to chicken oocyte membrane ex- tracts (Fig. 7). The concentrations of radiolabeled ligands in this experiment were chosen so that it was possible to perform all incubations with nitrocellulose strips containing identical pairs of bovine receptor/oocyte extract. All four types of lipoproteins were recognized by the bovine LDL receptor, which is known to behave as a 130-kDa protein under non- reducing conditions (31). As well, all of the radiolabeled lipoproteins bound selectively to a protein of 95 kDa present in octyl glucoside extracts of oocyte membranes. Under the incubation conditions, which for each lipoprotein were iden- tical for the bovine LDL receptor and oocyte membrane extracts, the oocyte protein bound the chicken lipoproteins clearly stronger than it bound LDL and VLDL of human origin. This is in agreement with the data in Figs. 5 and 6, which revealed low affinity of human LDL for the chicken receptor. In addition, these ligand blotting results, together with those of Fig. 5, strongly suggest that LDL and VLDL from both species are recognized by the same receptor on oocyte membranes. A second, weak and diffuse band in the upper part of the gel (lanes 2, 4, and 6) most likely represents receptor-unrelated nonspecific binding, as the appearance of this band was inconsistent from experiment to experiment and when present, was also observed in incubations contain- ing a 50-fold excess of the respective unlabeled ligand (data not shown).

Competitiue Binding Studies-The ligand specificity of the oocyte receptor was further investigated by the solid-phase filtration binding assay. Fig. 8 shows the results of competitive

rooster HDL (O), human LDL (O), and VLDL (A), or LDL (A) from estrogen-treated rooster; and Panel C, '*'I-LDL from estrogen-treated

absence of unlabeled LDL (A) or methyl-LDL (A) from estrogen- rooster (5 pg/ml; specific activity, 200 cpm/ng) in the presence or

treated rooster. The amount of receptor-bound radiolabeled ligand was determined by filtration as described under "Experimental Pro- cedures."

16844 Lipoprotein Receptors in Oocytes

binding studies with precipitated octyl glucoside extracts. Panels A and B demonstrate that both chicken LDL and VLDL, but not human LDL, effectively compete with lZ5I- chicken-LDL and -VLDL for binding to the oocyte receptor site. The inefficiency of human LDL to compete with radio- labeled chicken lipoproteins in the concentration range used is likely due to the low affinity of the mammalian LDL for the avian receptor (cf. Figs. 2,6, and 7; Table I). As expected, the presence of rooster HDL in the incubation mixtures had no inhibitory effect on the receptor binding of either "'1- chicken-VLDL or -LDL. Fig. 8C shows that reductive meth- ylation of chicken LDL obliterates its interaction with the oocyte receptor in that it was unable to compete effectively for the binding of 1251-chicken-LDL. At the low concentration (5 pg/ml) of '251-LDL used in Fig. 8C, unlabeled chicken LDL a t a concentration of 200 pg/ml completely displaced the radiolabeled ligand from the receptor.

Immunologic Analysis-The above experiments suggested that extensive similarity might exist between the bovine re- ceptor for LDL and the avian receptor for LDL and VLDL. To further test this possibility, we purified the bovine LDL receptor (32), raised polyclonal rabbit anti-receptor antibod- ies, and performed immunoblotting experiments. As shown in Fig. 9, the anti-bovine LDL receptor IgG specifically bound to a 95-kDa protein present in octyl glucoside extracts from chicken oocyte membranes. The bovine LDL receptor was visualized as a 130-kDa protein, its expected position of

200-

3 0 97- T

r' 68-

IgG: Immune Control FIG. 9. Immunoblotting of lipoprotein receptors with anti-

bovine LDL receptor IgG. Purified bovine LDL receptor (0.4 pg/ lane) and octyl glucoside extract of chicken oocyte membranes (35 pg protein/lane) were subjected to electrophoresis on 4.5-18% SDS- polyacrylamide gels under nonreducing conditions, followed by trans- fer to nitrocellulose and immunoblotting as described under "Exper- imental Procedures." Incubations contained 10 pg/ml of anti-bovine LDL receptor IgG (Immune) or nonimmune rabbit IgG (Control). To detect bound IgG, '%I-labeled goat anti-rabbit IgG (0.5 pg/ml; specific activity, -lo3 cpm/ng) was used. Exposure to XAR-5 film was for 12 h a t room temperature. M, standards are indicated.

migration in SDS-polyacrylamide gels under nonreducing conditions (31). Nonimmune rabbit IgG failed to show any reactivity (Fig. 9).

DISCUSSION

The present studies demonstrate that chicken oocytes pos- sess membrane receptor sites for the interaction with plasma lipoproteins of the low and very low density class, LDL and VLDL. The receptor was present in a membrane fraction prepared from ovarian follicles and could be extracted with the nonionic detergent octyl glucoside. This membrane frac- tion was derived from follicular tissue that contained, in addition to the oocyte plasma membrane, the noncellular basal lamina and perivitelline layer, and a single layer of granulosa cells, all of which lend rigidity to the structure of the oocyte and its plasma membrane (9). There is ample evidence that in oviparous species, which store large amounts of nutrients in their eggs, receptor-mediated endocytosis of hepatically derived yolk proteins takes place in the plasma membrane of the oocyte, and that other follicular cells are not themselves involved in yolk protein transport. For ex- ample, in the case of avian VLDL, elegant morphological studies by Gilbert and collaborators (9,11) have demonstrated that the lipoprotein particles freely cross the intergranulosa cell channels as well as the basal lamina and are then rapidly endocytosed after accumulating in coated regions of the oocyte plasma membrane. Furthermore, indirect evidence for the existence of specific receptors for VLDL on oocyte membranes has been provided by binding studies using lZ5I-labeled VLDL and oocyte membranes prepared essentially as described here (10,24). Recently, VLDL uptake and processing by the hen's oocyte has been demonstrated by analysis of radioactive in- traoocytic polypeptides following in vivo injection of VLDL containing 'H-labeled apolipoproteins (12). However, no at- tempts to identify and isolate the responsible receptor pro- tein(s) have been reported to date. In the current studies, we have employed detergent solubilization, a solid-phase binding assay, ligand blotting (31), and immunological analysis in order to characterize some of the properties of the chicken oocyte receptor for lipoproteins of the low and very low density class.

The chicken lipoproteins used in these experiments were fractions of pure VLDL ( p < 1.006), LDL (isolated at p = 1.025-1.063, and devoid of apolipoprotein VLDL-11), and HDL ( p = 1.15-1.21). The onset of egg-laying in the hen is accompanied by the dramatic estrogen-mediated induction of apolipoprotein VLDL-I1 synthesis and production of triglyc- eride-rich VLDL (491, with a concomitant drop in LDL levels. The function of apolipoprotein VLDL-I1 remains unknown; however, in this context it appears conceivable that the pres- ence of apolipoprotein VLDL-I1 on newly synthesized VLDL particles prevents lipolysis of the triglyceride moiety of VLDL and thus their conversion to lipoproteins of the low density class. Other possible roles of apolipoprotein VLDL-I1 in lip- oprotein metabolism are discussed below. Taken together, these observations suggest that VLDL, rather than LDL, is the physiologic ligand for a receptor involved in mediating the endocytosis of a nutrient lipoprotein into the oocyte. Nevertheless, because of the well-established role of the LDL receptor in mammalian cholesterol transport and metabolism (50) and the large amount of information on this protein, we were interested in the possible relationship of the avian oocyte receptor with its mammalian somatic counterpart.

We first adapted the methodology developed for the solu- bilization and measurement of LDL receptor activity (40) to the avian oocyte system. After solubilization with octyl glu-

Lipoprotein Receptors in Oocytes 16845

coside and dilution (40), the precipitated oocyte membrane extract bound both chicken LDL and VLDL with similar high affinity and capacity. Scatchard analysis of the binding data revealed a single binding site with a Ka of 10.1 pg/ml and 13.0 pg/ml for LDL and VLDL isolated from laying hen plasma, respectively. Lipoproteins prepared from estrogen-treated roosters bound with almost identical characteristics (Table I). In two previous studies that employed sedimentation bind- ing assays on crude membrane fractions (10,24), the reported Kd values were 12 pg/ml (10) and 43.5-45.5 pg/ml (24), respectively. Since the methodologies used in these two stud- ies were essentially identical, the reason for the discrepancy between the values obtained by the authors is not immediately apparent. However, the VLDL apoprotein content of the lipoproteins used in these studies cannot be evaluated, since their low molecular weight components were not shown (10). Secondly, these authors used crude membrane fractions from oocytes which are likely to be contaminated with adhering endogenous yolk proteins, lipoproteins in particular, which would interfere with the binding of radiolabeled LDL and VLDL. Here, we have used pure VLDL and LDL fractions (Fig. 1) and have greatly reduced the possibility of ligand contamination of receptor-containing samples by washing membranes extensively with aqueous buffers and subsequent extraction with octyl glucoside, followed by removal of re- maining soluble proteins after dilution of the octyl glucoside extract. This treatment leads to a significant enrichment of oocyte receptors, resulting in a specific activity of -70 pg/mg protein, compared to the reported values of 2 pg/mg (24) and 1.4 pg/mg (10) obtained with crude membranes. The receptor did not bind lZ5I-VLDL or -LDL when 20 mM EDTA was present in the incubation mixture, in agreement with previous data (9, 24), and in analogy to the mammalian LDL receptor (32, 40).

An additional property which the oocyte receptor shares with other plasma membrane receptors is its susceptibility to inhibition by the drug suramin (47). Ligand binding to the mammalian LDL receptor (47), the apolipoprotein E receptor (51), the locust oocyte receptor for vitellogenin (52) as well as to a receptor not involved in lipoprotein transport, that for platelet-derived growth factor (53) has been shown to be completely abolished by suramin. Furthermore, preformed ligand-receptor complexes can be dissociated with suramin. By the use of the solid-phase binding assay, we found that the drug has the same striking effect on the interaction of chicken VLDL with its receptor (Fig. 3). Suramin has been previously used to release pure lipoprotein receptors bound to ligand affinity matrices (47) and is hoped to be useful in the further characterization and purification of the oocyte recep- tor protein.

We have begun studies on the isolation of the oocyte receptor with its identification by ligand blotting (31), a technique that originally has been developed for visualization of LDL receptors in a variety of tissues and recently has been used in several modifications (54-56) in similar systems. During preliminary experiments we found that the receptor is extremely sensitive to proteolytic inactivation by Pronase; 50% of its activity is lost upon incubation with 1 pg/ml of the enzyme for 10 min at 37 "C (Fig. 4). This effect of Pronase is more pronounced than that on the LDL receptor from bovine adrenocortical membranes (40) or human fibroblasts (50). Thus, in order to successfully visualize the intact oocyte lipoprotein receptor by ligand blotting, octyl glucoside ex- tracts were prepared in the presence of a mixture of protease inhibitors. When such extracts were subjected to electropho- resis in SDS gels in the absence of disulfide-reducing agents

without prior heating, the receptor could be identified by incubation of nitrocellulose replicas of the gels with chicken lZ5I-VLDL or -LDL as a protein of apparent M, 95,000, determined by comparison with the position of migration of myosin, phosphorylase b, and bovine serum albumin. The chicken oocyte receptor is therefore significantly smaller than the mammalian LDL receptor, which migrates to the position of a protein with an apparent M, of 130,000 under nonreducing conditions (31).

In order to obtain further evidence that the 95-kDa protein indeed represented the high affinity receptor for chicken VLDL and LDL, we took advantage of our dual system for functional analysis, consisting of binding assay and ligand blotting. This strategy established the specificity of the recep- tor: chicken VLDL and LDL, and human LDL, but not human methyl-LDL, nor chicken HDL were able to interact with the chicken receptor. Furthermore, suramin completely inhibited binding of apolipoprotein B-containing lipoproteins to the 95- kDa oocyte receptor.

Reductively methylated human LDL has been shown to fail to bind to the LDL receptor due to the modification of lysine residues in apolipoprotein B essential for the interaction of the lipoprotein with its receptor (34, 47, 57). The fact that reductively methylated chicken LDL also does not bind to the oocyte receptor, as indicated by its inability to compete with the binding of lZ5I-chicken LDL (Fig. 8C), suggests that lysine residues are important for LDL binding in the avian system as well. While the current data indicate that apolipoprotein B plays a key role in oocyte receptor recognition, we do not know whether apolipoprotein VLDL-I1 participates directly or indirectly in this process as well. In quantitative electron microscopical studies, Perry et al. (11) observed similar Kd values for oocyte plasma membrane binding of lipoproteins from immature hens (presumably mainly LDL) and laying hens (VLDL). Since laying hen VLDL particles contain at least 20 times the number of apolipoprotein VLDL-I1 mole- cules per particle than do immature hen low density lipopro- teins (49, 58), these authors suggested that apolipoprotein B mediates the binding of both VLDL and LDL to a single receptor, in analogy to the mammalian receptor for LDL (59, 60). However, as outlined above, the physiological role, if any, of apolipoprotein VLDL-I1 has not been established, and a participation in oocyte receptor binding cannot be excluded. In particular, a function of apolipoprotein VLDL-I1 in mod- ulation of receptor binding in vivo or, alternatively, in direct- ing VLDL particles preferentially to oocytes, and not to somatic cells, must be considered. Investigations on the apo- protein specificity of the chicken oocyte lipoprotein transport system and the function of apolipoprotein VLDL-I1 are now in progress.

Ligand blotting experiments revealed extensive cross-spe- cies ligand binding capacity in that the bovine LDL receptor bound not only the human apolipoprotein B-containing lipo- proteins, LDL and VLDL, but also chicken LDL and VLDL with apparently little discrimination against the avian ligands (Fig. 7). To our knowledge, this is the first demonstration that nonmammalian lipoproteins are able to bind to mam- malian LDL receptors. Furthermore, as discussed above, the avian receptor bound not only chicken lipoproteins, but also human LDL and VLDL, albeit with low affinity (Table I). The competitive (Fig. 5) as well as the direct (Fig. 7) ligand blotting experiments also demonstrated that apolipoprotein B-containing lipoproteins from both species bound to the same 95-kDa protein present in octyl glucoside extracts of oocyte membranes.

Besides similar lipoprotein specificity, the mammalian LDL

16846 Lipoprotein Receptors in Oocytes

receptor and the chicken oocyte receptor share another prop- erty: in ligand blotting experiments, both the LDL receptor (31) and the chicken receptor (data not shown) fail to bind radiolabeled lipoproteins if electrophoresis had been per- formed in the presence of disulfide-reducing agents. This suggests that intrachain disulfide bonds within both receptor molecules are important for retention of their function follow- ing electrophoresis in the presence of SDS and transfer to nitrocellulose.

Finally, polyclonal antibodies directed against the purified bovine LDL receptor specifically recognized the 95-kDa oocyte receptor (Fig. 9), indicating extensive structural simi- larities between the receptors. If electrophoresis had been performed in the presence of sulfhydryl-reducing compounds, the polyclonal anti-receptor IgG reacted only very weakly with both the bovine and the chicken oocyte receptor in immunoblots (data not shown). Under these conditions, both immunoreactive proteins showed significantly decreased mo- bility on SDS-polyacrylamide gels. This behavior has been ascribed, in the case of bovine and human LDL receptors, to the unfolding of the above-mentioned intrachain disulfide bonds, predominantly located in the ligand binding region (31,61,62). Inasmuch as a difference in apparent M, of about 35,000 between the mammalian LDL receptor and the chicken oocyte receptor is observed under both reducing and nonre- ducing conditions, the structural basis for this difference will be the center of future investigation. Preliminary evj&n~. suggests that the chicken oocyte receptor is a glycoprotein containing sialic acid, and thus represents, if the analogy with the mammalian LDL receptor holds, the product of post- translational processing of its carbohydrate moiety (63, 64).

We do not know whether chicken somatic cells express lipoprotein receptor(s) with physiologic function(s) equivalent to that of their mammalian counterpart. It is intriguing to speculate that the 95-kDa protein is selectively expressed in chicken oocytes to mediate the uptake of triglyceride-rich, apolipoprotein B-containing particles. Studies to directly demonstrate the involvement of this protein not only in binding of VLDL, but also in endocytosis of the lipoprotein, are now underway.

Acknowledgments-We greatly appreciate the excellent technical assistance of Rita Langford. We thank Beverly Bellamy for expert assistance in preparation of this manuscript. We are grateful to Lilydale Poultry Sales, Edmonton, for permission to collect oocytes during slaughter.

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