Turnover of the surface proteins and the receptor for serum asialoglycoproteins in primary cultures of rat hepatocytes.

R Warren,D Doyle

doi:10.1016/s0021-9258(19)69971-0

Abstract

The turnover of plasma membrane proteins in primary rat hepatocyte cultures was examined by following the loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination using 125I and 131I. Most plasma membrane proteins had similar rates of degradation, having a half-life of approximately 85 h. By in situ labeling via lactoperoxidase-catalyzed iodination, as well as metabolically labeling cells with L-[35S]methionine, the asialoglycoprotein receptor, a plasma membrane constituent, was identified and shown to exist in three forms which were structurally related. The turnover of receptor on the cell surface was examined by following the loss of iodinated cell surface receptor, while the turnover of total cellular receptor, including both surface and internally localized receptor was assayed by following the loss of receptor labeled metabolically with [35S]methionine. The turnover rate in both cases was approximately 20 h. Receptor-mediated endocytosis of asialoglycoproteins had no effect on the turnover of the plasma membrane proteins or receptor. Based on estimates of the rate of metabolism of the asialoglycoprotein ligand relative to the turnover rate of the receptor, we conclude each molecule of receptor can deliver about 1,000 molecules of ligand to the lysosome to be degraded.

Highlights

MATERIALS AND METHODSChurniculs-Except where indicated,chemicals used were reagent grade from commercial suppliers.Acrylamide and ,V,N‘-dimethyIhisacrylamide were obtained from Eastman
Diated endocytosis of asialoglycoproteins had no effect In this manuscript, we have examined the turnover of the on the turnover of the plasma membrane proteins or externally oriented plasma membraneproteins inprimary receptor
After of the asialoglycoprotein ligand relative to the turnover rate of the receptor, we conclude each molecule of receptor can deliver about 1,000 molecules of ligand to the lysosome to be degraded

Summary

MATERIALS AND METHODS

Churniculs-Except where indicated,chemicals used were reagent grade from commercial suppliers.Acrylamide and ,V,N‘-dimethyIhisacrylamide were obtained from Eastman. Peptide mappingby proteolysis in sodium dodecyl sulfate-containpowder by the method describedby Hudgin et af.[21], except for the ing po1,vacrylamide gels wasperformedessentially as described bv following modifications: the pellet derived from the acetawteash was Cleveland et al [31] with the following modifications: following oneextracted twice with Triton X-100 instead of once ("acetone powder dimensional electrophoresis in 97 polyacrylamide, the entire lane of detergent extract").No significant activitywas extracted upon further interestwascut o u t andrinsed in 0.125 M Tris/HCI,pH 6.8, 0.1'; Turnover of PSruortfeaicnes in Hepatocytes. '%Labeled material was counted in a Beckman LS-7000 scintilla- Hepatocyteswere iodinatedas described under "Materialsand Methtion counter by first dissolving the sample in 1.0 ml of Protosol (New ods." The cell plates were washed with Hanks' bufferf,ollowed by the England Nuclear) for 30 min at 45°C. followed by the addition of 10 addition of fresh medium, and reincubated at 37°C. At the time points designated, the cells were prepared for the determination of acid-insoluble radioactivity as described under

RESULTS

IO 20 30 40 50

Findings

DISCUSSION