Site-directed mutagenesis of amino acids in the cytoplasmic loop 6/7 of Na,K-ATPase.
Ann N Y Acad Sci
; 986: 96-100, 2003 Apr.
Article
en En
| MEDLINE
| ID: mdl-12763780
The loop between transmembrane helices 6 and 7 (L6/7) of P-type ATPases has been suggested to be important for the functional linkage of ion binding and enzyme phosphorylation or to be a site of initial cation binding. To investigate the role of L6/7 in Na,K-ATPase, alanine substitutions were made for charged and conserved residues in L6/7 of the human alpha1 subunit and the proteins were expressed in yeast for analysis. All mutants except the triple mutant E825A/E828A/D830A bound ouabain. Although the equilibrium dissociation constant for ouabain binding by most mutants was similar to the wild-type value, the K(d) of R837A for ouabain binding was approximately 15-fold higher than the wild-type K(d). (18)O exchange measurements indicated that the apparent affinity of this mutant for Pi was reduced about 3-fold. The concentration dependence of KCl inhibition of ouabain binding or of NaCl inhibition of ouabain binding revealed 2-4-fold changes in the apparent affinity for cations in the E825A, E828A, and R837A mutants. The E825A and E828A mutants lost the ability to bind ouabain after extraction with 0.1% SDS or after brief heating, indicating that these mutations affected the stability of the enzyme. The ATPase activity of the other mutants was measured after extraction of crude yeast membranes with 0.1% SDS. For all mutants except R834A, R837A, and R848A, the activity was at least 50% of wild-type activity.
Buscar en Google
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ATPasa Intercambiadora de Sodio-Potasio
Límite:
Humans
Idioma:
En
Revista:
Ann N Y Acad Sci
Año:
2003
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos