Binding of apolipoprotein A-I and A-II after recombination with phospholipid vesicles to the high density lipoprotein receptor of luteinized rat ovary.

J Hwang,K M Menon

doi:10.1016/s0021-9258(18)89074-3

Abstract

To determine the apolipoprotein specificity of high density lipoprotein (HDL) receptor, apolipoprotein A-I (apo-AI) and apolipoprotein A-II (apo-AII) purified from high density lipoprotein3 (HDL3) were reconstituted into dimyristoyl phosphatidylcholine vesicles (DMPC) and their ability to bind to luteinized rat ovarian membranes was examined. Both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC were shown to bind to ovarian membranes with Kd = 2.87 and 5.70 micrograms of protein/ml, respectively. The binding of both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC was inhibited by unlabeled HDL3, apo-A-I.DMPC, apo-A-II.DMPC, apo-C-I.DMPC, apo-C-II.DMPC, apo-C-III1.DMPC, and apo-C-III2.DMPC, but not by DMPC vesicles, bovine serum albumin.DMPC or low density lipoprotein. Since the binding labeled apo-A-I.DMPC and apo-A-II.DMPC was inhibited by the DMPC complexes of apo-C groups, the direct binding of 125I-apo-C-III1.DMPC was also demonstrated with Kd = 9.6 micrograms of protein/ml. In addition, unlabeled apo-A-I.DMPC, and apo-A-II.DMPC, as well as apo-C.DMPC, inhibited 125I-HDL3 binding. 125I-apo-A-I, 125I-apo-A-II, and 125I-apo-C-III1 in the absence of DMPC also bind to the membranes. These results suggest that HDL receptor recognizes apolipoprotein AI, AII, and the C group and that the binding specificity of the reconstituted lipoproteins is conferred by their apolipoprotein moiety rather than the lipid environment. In vivo pretreatment of rats with human chorionic gonadotropin resulted in an increase of 125I-apo-A-I.DMPC, 125I-apo-A-II.DMPC, and 125I-apo-C-III1.DMPC binding activities. However, no induction of binding activity was observed when the apolipoprotein was not included in DMPC vesicles. An examination of the equilibrium dissociation constant and binding capacity for 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC after human chorionic gonadotropin treatment revealed that the increase in binding activity was due to an increase in the number of binding sites rather than a change in the binding affinity. These results further support our contention that apo-A-I, apo-A-II, and the apo-C group bind to HDL receptor. In conclusion, the HDL receptor of luteinized rat ovary recognizes apolipoproteins A-I, A-II, and the C group but not low density lipoprotein, and the binding is induced by human chorionic gonadotropin in vivo.

Highlights

(DMPC)and theirability tobind to luteinized rat ovarian membranes was examined
11-DMPC was inhibited by the DMPC complexes of recognize bothapo-Bandapo-E [12,13,14].,which apo-C groups, thdeirecbtinding of '261-apo-C- apolipoprotein is recognized by HDL receptor is not well
Since HDL3contains two majqr membranes. These results suggest that HDL receptor apolipoproteins, apo-A-I andapo-A-11, experiments were carrecognizes apolipoprotein AI, AII,and theC group and ried out to purify apo-A-I and apo-A-11 from human HDL3, that the binding specificity of the reconstituted lipo- followedby an examination of their ability to bind to rat proteins is conferred by their apolipoprotein moiety ovarian membranes

Summary

ApolipoprSopteicnificity oOf uRaartian

HDL Receptor monomer standard protein powder was obtained from Miles Laboratories, Inc. The relative purification of membrane preparations was ascertained by assaying 5'-nucleotidase activity. The extent of mitochondrial, lysosomal, and microsomal contamination was checked by assaying specific marker enzymes [31]. Membranes were prepared on the same day as Methods the binding assay. The incubations usually were carried out in a total and HDL, (cf, 1.125-1.210 g/ml) were fractionated by sequential volume of 0.25 ml of 25 mM Tris-HC1 buffer, pH 7.4, containing "'I-. Ti rotor, using KBr andNaCl for density adjustment as described by pg of protein/ml; specific activity for '251-apo-A-I,1251-apo-A-IaI,nd. Free I w I ion was removed from the "'I-apo- precooled buffer (25 mM Tris, pH 7.4) to each tube followed by. DMPC, and 125Z-Apo-C-IIZl.DMPC-DMPCvesicleswere prepared by the procedure of Roth et al [26]. The solution was sonicated for 20 min at 30 "C using a Sonifier Cell Disruptor, Model

RESULTS

Apolipoprotein Specificity of Rat OvariaHnDL Receptor

Unlabeled substance"

DISCUSSION

Apolipoprotein Specificityof Rat Ovarian HDL Receptor