Quantification of rat hepatocyte transferrin receptors with poly- and monoclonal antibodies and protein A.
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 conjunction with the less damaging of two batches of the enzyme, 129,000 receptors were found per cell, with 47,000 (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 per 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 microM) produced a 32%-50% shift of Tf-R from the surface to the inside, whereas short-term incubation with epidermal growth factor (0.17 mM) brought about no clear-cut Tf-R redistribution.
Rudolph
J R
JR
Department of Pathology, McMaster University Health Sciences Centre, Hamilton, Ontario, Canada.
Regoeczi
E
E
Southward
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D008099:Liver / Q000032:analysis* / Q000166:cytology / Q000187:drug effects; D008985:Monensin / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011990:Receptors, Transferrin / Q000032:analysis* / Q000276:immunology; D013205:Staphylococcal Protein A; D014168:Transferrin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011990:Receptors, Transferrin; D013205:Staphylococcal Protein A; D014168:Transferrin; D004815:Epidermal Growth Factor; D008985:Monensin