Rat ceruloplasmin (rCp) has been labeled with rhodamine B isothiocyanate (RBITC). RBITC-labeled rCp (RBITC-rCp) was tested as a probe for ceruloplasmin receptors on rat erythrocytes using fluorescence microscopy. Film negatives of blood smears were analyzed by microdensitometry to give relative optical densities for the amount of RBITC-rCp bound on the plasma membrane. Binding of RBITC-rCp to erythrocytes and platelets was saturable, reversible, and specific as only rCp could displace it. In both cases trypsin-treated cells showed greatly reduced binding. Applying the same technique to rat lung tissue, bronchial muscle demonstrated evidence of a receptor. / An electrophoresis mobility shift assay (EMSA) was developed to study the binding of rCp to its erythrocyte receptor. $\sp{125}$I-rCp, native polyacrylamide gel electrophoresis (PAGE), and detergent-solubilized plasma membrane were required. Triton X-100 extracted receptor bound apo-rCp as well as native rCp and had a M$\sb{\rm r}$ of 150,000. The binding of $\sp{125}$I-rCp was specific as only rCp could displace it. The receptor extracted with SDS detergent showed activity after 80$\sp\circ$C for 10 min, so SDS-PAGE was used to separate the membrane proteins. Three classes of binding proteins were detected: (1) nonspecific irreversible, (2) nonspecific reversible, and (3) specific and reversible--the receptor. Using this technique the M$\sb{\rm r}$ for the receptor was less than 80,000. / The reason chloroethylamine-treated agarose binds Cp from plasma preferentially has been elucidated. Polyethylenimine tentacles are formed which require a uniform negative surface charge for proteins to bind. / Source: Dissertation Abstracts International, Volume: 53-07, Section: B, page: 3461. / Major Professor: Earl Frieden. / Thesis (Ph.D.)--The Florida State University, 1992.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_76697 |
| Contributors | Stern, Robert Victor., Florida State University |
| Source Sets | Florida State University |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | 138 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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