Effects of incorporated trypsin on anion exchange and membrane proteins in human red blood cell ghosts

Abstract

Varying concentrations of trypsin were sealed into human red cell ghosts and the effects on membrane proteins and sulfate equilibrium exchange were studied. After incubation for 45 min at 37 °C, pH 7.2, the following observations were made: above 10 ng/ml the ghosts undergo fragmentation without lysis. Dodecyl sulfate gel electrophoresis shows that the digestion of spectrin and of the protein in band 2.1 (nomenclature of Steck (1974) J. Cell. Biol. 62, 1–19) is nearly complete at 50 ng/ml, that of the protein in band 3 at 25 μg/ml. After digestion at 25 μg/ml, about 60% of the total protein of the membrane is released and the original bands of conventional dodecyl sulfate gel electropherograms of the remaining protein are nearly completely abolished. In their place three new bands appear representing peptides with molecular weights of 58 000, 48 000 and 34 000, respectively. Sometimes a fourt peptide with a molecular weight of approx. 13 000 is also observed. Using a radioactive labeling technique it is shown that the two peptides with the highest molecular weights are derived from the protein in band 3. Labeling with diazo[ 3 5S]sulfanilic acid indicates that in addition to the peptides in the described four Coomassie blue-stainable bands, other peptides with molecular weights up to 100 000 are still present in the exhaustively trypsinized ghosts. External trypsin has no effect on the sulfate equilibrium exchange in ghosts while internal trypsin causes inhibition. Inhibition becomes apparent at trypsin concentrations exceeding those required to produce a complete digestion of spectrin. It remains incomplete, even at the highest intracellular concentrations which cause maximal effects on all membrane proteins, including the protein in band 3. Under these conditions strong further inhibition can be produced by agents which are known to inhibit anion transport in untreated red cells and ghosts. These agents include the penetrating 1-fluoro-2,4-dinitrobenzene and the nonpenetrating phlorizin, 4-acetamido-4′-isothiocyanato stilbene-2,2′-disulfonic acid, 4,4′-diacetamido stilbene-2,2′-disulfonic acid, and 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB). Unlike the other nonpenetrating inhibitors APMB is not only capable of inhibiting at the outer but also at the inner membrane surface. Treatment with internal trypsin does not significantly reduce the inhibition by incorporated APMB. The described observations suggest that after exhaustive tryptic digestion of the major membrane proteins, the receptor sites for typical inhibitors of anion transport continue to exert their function.