Topology of the C-terminus of sodium hydrogen exchanger isoform-1: presence of an extracellular epitope.
In this study, the topology of the C-terminus of Na(+)/H(+) exchanger-1 (NHE-1) was examined using red blood cell (RBC) membrane vesicles. Specific polyclonal antibodies were raised against the C-terminus of the rat NHE-1 using a glutathione S-transferase fusion protein antigen. Enzyme-linked immunosorbent assay (ELISA) and flow cytometer analyses were employed. NHE-1 antibodies recognized a 110-kDa protein in the rats and mice, but not in the humans. RBC vesicles were resealed using 1 mM MgCl(2), and the right side out (RSOVs) and the inside out vesicles (ISOVs) were selected by concanavalin A. NHE-1 antibodies reacted strongly with the RSOVs and poorly with the ISOVs in an ELISA. The reaction with the unsealed vesicles was stronger than the resealed RSOVs. In contrast, the monoclonal alpha-actin antibodies reacted poorly with the RSOVs, but extremely strongly with the ISOVs. Flow cytometer analysis also showed a strong reaction of NHE-1 antibodies with the intact and permeabilized rat RBCs. The antibodies raised in this study also contain a population which reacts with the internal epitope in the NHE-1 C-terminus. These findings suggest that the NHE-1 C-terminus contains epitope(s) exposed extracellularly.
Khan
I
I
Department of Biochemistry, Faculty of Medicine, Kuwait University, Kuwait. Islam@hsc.kuniv.edu.kw
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D005434:Flow Cytometry; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; D000939:Epitopes; D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1