Quantification of rat hepatocyte transferrin receptors with poly- and monoclonal antibodies and protein A

Publisher Website
Google Scholar
Abstract
The content and distribution of transferrin receptors (Tf-R) in suspended adult rat hepatocytes were studied using 125I-protein A in combination with either a monoclonal (MRC OX-26) or a polyclonal antibody to Tf-R. Internal receptors were made accessible by permeabilization with digitonin. The number of Tf-R detected depended on the batch of collagenase used for liver perfusion. By using the monoclonal reagent in conjction with the less damaging of two batches of the enzyme, 129000 receptors were found per cell, with 47000 (37%) of these on the surface. The polyclonal reagent yielded Tf-R numbers which were consistently higher than those obtained with MRC OX-26. This difference is interpreted as being due to the binding of several (on the average 5–6) molecules of polyclonal IgG pen molecule of Tf-R. Remarkably, transferrin binding by Tf-R was not affected by this cluster of associated IgG and the overlayer of protein A. Parallel studies with 131I-transferrin in a simplified binding assay system yielded surface Tf-R estimates which, in most cases, were close to the values obtained with MRC OX-26. After prolonged exposure to collagenase, the ligand-binding capacity of Tf-R was more affected than its immunoreactivity. In preliminary studies, monensin (10 μM) produced a 32%–50% shift of Tf-R from the surface to the inside, whereas short-term meubation with epidermal growth factor (0.17 mM) brought about no clear-cut Tf-R redistribution.