Characterization of the prolactin receptor in cell fractions from rat liver.

[125I]Prolactin (PRL) was covalently cross-linked to its binding sites in subcellular fractions of female rat livers using NHSAB and UV irradiation. Analysis by non-reducing SDS-PAGE showed that all fractions with specifically bound radioactive hormone contained a major autoradiographic band (eliminated with unlabeled PRL) of similar electrophoretic mobility consistent with a MW of 36K for the receptor. In addition, microsomal membranes were treated with a zwitterionic detergent (CHAPS), solubilizing 30-60% of the specifically bound radioactivity.

Cross-linking of hemoglobin to the cytoplasmic surface of human erythrocyte membranes. Identification of band 3 as a site for hemoglobin binding in Cu2+-o-phenanthroline catalyzed cross-linking.

The addition of hemoglobin to isolated membrane ghosts of human erythrocytes followed by catalytic oxidation with Cu2+-o-phenanthroline results in the covalent attachment of hemoglobin to the membrane. A decrease in the mobility of Band 3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to an increase in molecular weight of approximately 16,000 suggests that Band 3 is a site for the covalent attachment of hemoglobin to the membrane under these conditions. This conclusion was confirmed using two-dimensional gels in which the covalent linkage was cleaved with 2-mercaptoethanol in the second dimension.