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(Hypertension. 1995;25:694-698.)
© 1995 American Heart Association, Inc.

Articles

Mutational Inactivation of the Catalytic Domain of Guanylate Cyclase-A Receptor

Zhen-Hua Miao; Dong-Li Song; Janice G. Douglas; Chung-Ho Chang

From the Departments of Medicine and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio; and the Department of Medical Genetics, University of Toronto, Ontario, Canada (D.-L.S.).

Correspondence to Dr Chung-Ho Chang, Department of Medicine, Division of Hypertension, Case Western Reserve University School of Medicine, W 165, Cleveland, OH 44106.

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.

Key Words: guanylate cyclase • mutation • receptors, atrial natriuretic factor

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