Effects of pH and temperature on the interaction of an impermeant probe with surface proteins of the human red blood cell.
The conformation of the outer surface of the human red cell membrane has been studied under various conditions using the impermeant probe [125I]diazodiiodosulfanilic acid. At least seven polypeptides were labeled by the reagent, including the three extractable glycoproteins separable by the electrophoretic method employed. The Mr = 43,000 protein band was shown to contain two labeled species, one a glycoprotein, in addition to its major constituent, red cell actin. The extent and pattern of labeling were very sensitive to changes in pH and temperature. Total labeling increased with increasing pH and was greater at 4 degrees C than 37 degrees C. Binding of the probe to the Mr = 90,000 polypeptide and the major glycoprotein were relatively increased with increasing pH and temperature while opposite effects were observed for the Mr = 43,000 peptide(s). The pH effects on external membrane labeling were rapidly reversible. Results were similar in cells of different densities, suggesting that the pH and temperature effects were not related to cell age. The data presented emphasize the lability of membrane conformation and reactivity and thus the necessity to consider carefully the conditions of labeling in interpretation of studies using impermeant probes.
Effects of pH and temperature on the interaction of an impermeant probe with surface proteins of the human red blood cell. The conformation of the outer surface of the human red cell membrane has been studied under various conditions using the impermeant probe [125I]diazodiiodosulfanilic acid. At least seven polypeptides were labeled by the reagent, including the three extractable glycoproteins separable by the electrophoretic method employed. The Mr = 43,000 protein band was shown to contain two labeled species, one a glycoprotein, in addition to its major constituent, red cell actin. The extent and pattern of labeling were very sensitive to changes in pH and temperature. Total labeling increased with increasing pH and was greater at 4 degrees C than 37 degrees C. Binding of the probe to the Mr = 90,000 polypeptide and the major glycoprotein were relatively increased with increasing pH and temperature while opposite effects were observed for the Mr = 43,000 peptide(s). The pH effects on external membrane labeling were rapidly reversible. Results were similar in cells of different densities, suggesting that the pH and temperature effects were not related to cell age. The data presented emphasize the lability of membrane conformation and reactivity and thus the necessity to consider carefully the conditions of labeling in interpretation of studies using impermeant probes.