Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin.
The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.
Ottnad
E
E
Department of Medicine, University of Heidelberg, Federal Republic of Germany.
Via
D P
DP
Sinn
H
H
Friedrich
E
E
Ziegler
R
R
Dresel
H A
HA
D000107:Acetylation; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051381:Rats; D017457:Receptors, Albumin; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*
D008024:Ligands; D008077:Lipoproteins, LDL; D008565:Membrane Proteins; D017457:Receptors, Albumin; D011956:Receptors, Cell Surface; D011973:Receptors, LDL; D012710:Serum Albumin, Bovine