ATP-dependent inactivation and reactivation of constitutively recycling galactosyl receptors in isolated rat hepatocytes.
Isolated rat hepatocytes depleted of ATP with NaN3 without ligand lose galactosyl (Gal) receptors from the cell surface and accumulate inactive receptors within the cell [McAbee, D. D., & Weigel, P. H. (1987) J. Biol. Chem. 262, 1942-1945]. Here, we describe the kinetics of receptor redistribution and inactivation after ATP depletion with NaN3 and of receptor redistribution and reactivation after ATP recovery. Only intact cells (greater than 98% viable) isolated from Percoll gradients were assayed. Gal receptor activity and protein were measured by the binding of 125I-asialoorosomucoid (125I-ASOR) and 125I-anti-Gal receptor IgG (125I-IgGR), respectively, at 4 degrees C. Surface and total (surface and intracellular) cellular Gal receptors were measured in the absence or presence, respectively, of digitonin. Following ATP depletion, 60-70% of Gal receptor activity and protein were lost from cell surfaces with first-order kinetics (t1/2 = 6.5 min, k = 0.107 min-1) at an initial rate of 11,000 125I-ASOR binding sites cell-1 min-1. Lost cell-surface Gal receptors were transiently recovered still active inside the cell. After a short lag, total cellular receptor inactivation then proceeded with first-order kinetics (t1/2 = 13 min, k = 0.053 min-1) at an initial rate of 14,000 125I-ASOR binding sites cell-1 min-1. Up to half of all cellular Gal receptors were inactivated by 40 min. 125I-IgGR binding to NaN3-treated, permeable cells, however, was virtually constant. The distribution of total cellular receptors changed from 35% on the cell surface initially to 10% after 40 min of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
McAbee
D D
DD
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Weigel
P H
PH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D005690:Galactose / Q000378:metabolism*; D007118:Immunoassay; D007074:Immunoglobulin G; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*
D007074:Immunoglobulin G; D011956:Receptors, Cell Surface; C039707:galactose receptor; D000255:Adenosine Triphosphate; D005690:Galactose