Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA.

In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5'-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3'-untranslated region of 924 bp.

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.

The preparation of hydrophilic derivatives of band 3 protein by acylation of the human red blood cell membrane.

In situ reaction of erythrocyte membranes with dicarboxylic anhydrides leads to solubilization of hydrophobic integral proteins. Removal of peripheral proteins and bulk lipid by appropriate sedimentation and dialysis steps yields hydrophilic band 3 protein derivatives. These acyl compounds display size heterogeneity upon gel filtration.

Inhibition of anion transport across the red cell membrane by dinitrophenylation of a specific lysine residue at the H2DIDS binding site of the band 3 protein.

The inhibition of anion transport by dinitrophenylation of the red cell membrane is brought about by the modification of a single lysine residue located on the 17-kDa segment of the band 3 protein. This residue is identical with Lys a, which is also capable of reacting with one of the two isothiocyanate groups of 4,4'-diisothiocyano dihydro-stilbene-2,2'-disulfonate (H2DIDS). The rate of reaction between Lys a and 1-fluoro-2,4-dinitrobenzene is reduced when a second lysine residue on the 35-kDa segment of the band 3 protein becomes dinitrophenylated.

Senescent cell antigen is immunologically related to band 3.

IgG autoantibodies in human serum selectively bind to a glycopeptide antigen that appears on senescent and damaged cells in situ. We identified the membrane protein from which the senescent cell antigen is derived by using a phagocytosis-inhibition assay and immunoautoradiographic gel staining and electroblotting techniques. Results of the phagocytosis-inhibition assay revealed that only the purified transmembrane glycoprotein designated "band 3" and senescent cell antigen inhibited the phagocytosis of erythrocytes induced by IgG eluted from senescent erythrocytes.