Mutational inactivation of the catalytic domain of guanylate cyclase-A receptor.
Hypertension
; 25(4 Pt 2): 694-8, 1995 Apr.
Article
en En
| MEDLINE
| ID: mdl-7721418
ABSTRACT
Guanylate cyclase-A, the receptor for atrial natriuretic factor, contains a protein kinase-like domain and a catalytic domain in the intracellular region. To investigate the active site (the catalytic cavity) of guanylate cyclase-A, we amplified the catalytic domain plus three amino acids from the kinase-like domain of guanylate cyclase-A (GC-c) with polymerase chain reaction (PCR) and expressed it in Escherichia coli. During the screening of the PCR-cloned gene products with guanylate cyclase assay, a mutant that lacks enzyme activity was identified. Results of cDNA sequencing revealed that Leu 817 was replaced by an Arg residue in the mutated protein. The mutated GC-c bound to GTP-agarose as well as the wild-type protein, indicating that the binding capability of mutated GC-c to GTP is not significantly affected by the Arg substitution. Gel-filtration column chromatography showed that, like the wild-type GC-c, the mutated protein also formed a high-molecular-weight complex. Since mutation of Leu 817 to Arg abolishes the catalytic activity, Leu 817 is likely located near the active site of guanylate cyclase-A. These results demonstrate that the carboxyl fragment of guanylate cyclase-A is an ideal system for studying the active site of guanylate cyclase-A.
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Bases de datos:
MEDLINE
Asunto principal:
Receptores del Factor Natriurético Atrial
/
Guanilato Ciclasa
/
Mutación
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Hypertension
Año:
1995
Tipo del documento:
Article
País de afiliación:
Estados Unidos