title: The ATP-binding site of the erythrocyte membrane Ca2+ pump. Amino acid sequence of the fluorescein isothiocyanate-reactive region.
abstract: The erythrocyte plasma membrane Ca2+-pumping ATPase is known to form an acyl-phosphate catalytic intermediate, but there is otherwise little structural information linking it to the other mammalian ion-pumping ATPases which also form phosphorylated intermediates (the Na+, K+-ATPase of plasma membranes, the Ca2+-ATPase of sarcoplasmic reticulum, and the H+, K+-ATPase of gastric mucosa). We show here that this enzyme possesses a fluorescein isothiocyanate-reactive region similar to that possessed by these other ATPases. Low concentrations (10 microM) of fluorescein isothiocyanate inhibit the ATPase activity of this pump, and this inhibition is prevented by 4 mM ATP. ATP also inhibits the reaction of fluorescein isothiocyanate with a single amino acid residue on the 138-kDa polypeptide chain. A tryptic fragment containing the fluorescein-conjugated residue was isolated by high pressure liquid chromatography. The sequence of this peptide was determined to be NH2-Met1-Tyr2-Ser3-Lys4-Gly5-Ala6-Ser7-Glu8++ +-Ile9-Ile10-Leu11-Arg12-COOH; fluorescein isothiocyanate reacts with the lysine residue. The identities of residues 4-8 are the same as those in a sequence common to the other ATPases mentioned above, except that serine-7 of this sequence is changed to a proline in those ATPases. This substitution, sometimes not considered a homologous one, is not expected to have a major effect on the secondary structure or polarity of this region. Outside of this 5-residue core region of the fluorescein isothiocyanate-reactive site, the homologies among the different ion-pumping ATPases are limited.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000097:blood*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000097:blood*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D009928:Organ Specificity; D011485:Protein Binding; D013861:Thiocyanates
chemical_list: D005452:Fluoresceins; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate