Isolation and characterization of the rainbow trout erythrocyte band-3 protein

Rainbow trout (Salmo gairdneri) band-3 protein was isolated from trout erythrocyte plasma membranes by a combination of preparative SDS/PAGE and electroelution. High purity and recovery of the plasma membranes were achieved by a new method. This was demonstrated using 4,4'diiso-thiocyano[3H2]dihydro-stilbene 2,2'disulfonic acid (3H2DIDS) which specifically labels band-3 protein. On SDS/PAGE, band-3 protein yields a similarly diffuse pattern, as does mammalian band-3 protein, with an apparent Mr of 116,000. In situ chymotryptic cleavage/cross-linking experiments with 3H2DIDS reveal that the fragments cross-link as in human and mouse band-3 proteins but that there are minor differences. Treatment of trout erythrocytes with trypsin results in cleavage of the band-3 protein. Purified polyclonal antibodies raised against trout band-3 protein react with trout band-3 protein and do not crossreact with mouse or human band-3 protein. They react specifically with only one chymotryptic fragment of trout band-3 protein.