Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors.
To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.
Chappell
D A
DA
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Fry
G L
GL
Waknitz
M A
MA
Berns
J J
JJ
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism*; D001057:Apolipoproteins E / Q000378:metabolism; D002413:Cations, Divalent; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007536:Isomerism; D007700:Kinetics; D011973:Receptors, LDL / Q000378:metabolism*
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D002413:Cations, Divalent; D007457:Iodine Radioisotopes; D011973:Receptors, LDL