Mouse macrophage receptor for acetylated low density lipoprotein: demonstration of a fully functional subunit in the membrane and with purified receptor.
The functional molecular mass of the macrophage receptor for acetylated low density lipoprotein (Ac-LDL) was determined in membranes by radiation inactivation analysis. Membranes from tumors induced by the mouse macrophage cell line P388D1 were frozen and irradiated with high-energy electrons. Residual binding activity indicated a minimum functional molecular mass of 35,000 Da, considerably smaller than the active 260,000 M(r) protein seen on ligand blots under nonreducing conditions. Scatchard analysis of receptor binding gave no evidence of partially inactivated molecules. The receptor protein, purified by affinity chromatography and preparative gel electrophoresis, was incubated with dithiothreitol (0.1-100 mM) and retested for binding activity. Active subunits of 158,000 and 80,000 M(r) could be demonstrated by ligand blotting, with quantitative conversion of binding activity to the 80,000 M(r) species at 10 mM dithiothreitol. At 100 mM dithiothreitol, all binding activity was lost. Further size reduction was not detected by silver staining. These data suggest that the isolated mouse macrophage Ac-LDL receptor is a trimer with one class of SH groups involved in trimerization and another in the actual binding site. The monomeric species is fully active in vitro under mild reducing conditions. The radiation inactivation data also suggest that each monomeric unit is fully active and capable of functioning independently in the binding of ligands in the membrane.
Via
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DP
Department of Medicine, Baylor College of Medicine, Houston, TX.
Kempner
E S
ES
Pons
L
L
Fanslow
A E
AE
Vignale
S
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Smith
L C
LC
Gotto
A M
AM
Dresel
H A
HA
D000818:Animals; D015815:Cell Adhesion Molecules; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011973:Receptors, LDL / Q000302:isolation & purification / Q000378:metabolism*; D051116:Receptors, Scavenger
D015815:Cell Adhesion Molecules; D006882:Hydroxyapatites; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D051116:Receptors, Scavenger; C032629:acetyl-LDL; D017886:Durapatite