Hydroxylamine treatment differentially inactivates purified rat hepatic asialoglycoprotein receptors and distinguishes two receptor populations

Abstract

We previously showed that two subpopulations of asialoglycoprotein receptors (ASGP-Rs), designated State 1 and State 2 ASGP-Rs, are present in intact cells and that State 2 ASGP-Rs can be inactivated in permeable rat hepatocytes in a temperature- and ATP-dependent manner. These inactivated ASGP-Rs can be quantitatively reactivated by the addition of palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). Here we show that approximately 50% of purified rat ASGP-Rs are inactivated by treatment with hydroxylamine under mild conditions. The activity of affinity-purified ASGP-Rs was assessed by measuring the specific binding of 125I-asialo-orosomucoid (ASOR) in a dot-blot assay after immobilization onto nitrocellulose. Treatment of ASGP-Rs in solution with 0.0125-1.0 M NH2OH, pH 7.4, at 4 degrees C for 4 h resulted in a progressive loss of ASOR binding activity. ASGP-R inactivation with NH2OH occurred more readily at basic pH or at room temperature. Similar treatment with Tris had no effect on ASGP-R activity. The kinetics of ASGP-R activity loss and the dose-response for this inactivation were both biphasic, indicating the presence of two equal populations of ASGP-Rs with different sensitivities to NH2OH. The more sensitive population of ASGP-Rs (approximately 50%) was inactivated by treatment with 0.2 M NH2OH (4 degrees C, 4 h) or with 1.0 M NH2OH (4 degrees C, 1 h) without detectable peptide cleavage as assessed by SDS-polyacrylamide gel electrophoresis. State 1 ASGP-Rs, purified from chloroquine- or monensin-treated hepatocytes, showed significantly less sensitivity to NH2OH treatment (both in kinetics and dose dependence). Furthermore, under mild conditions NH2OH caused dissociation and inactivation of approximately 50% of the total ASGP-Rs (State 1 and State 2) that were prebound to ASOR-Sepharose, whereas the same treatment caused dissociation of only < 20% of State 1 ASGP-Rs from such preformed complexes. As shown in the accompanying paper (Zeng, F. Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387) all three RHL subunits of active ASGP-Rs, in fact, contain covalently attached palmitate and stearate. In cultured cells, [3H]palmitic acid is metabolically incorporated into all three subunits. These radiolabeled fatty acids are completely released from purified ASGP-Rs by mild NH2OH treatment.(ABSTRACT TRUNCATED AT 400 WORDS)