The carboxyl region contains the catalytic domain of the membrane form of guanylate cyclase.
A polypeptide containing the catalytic domain of an atrial natriuretic peptide receptor guanylate cyclase has been produced using a bacterial expression system. A carboxyl fragment of the membrane form of guanylate cyclase from rat brain, which contains a region homologous to soluble guanylate and adenylate cyclases, was expressed in Escherichia coli with a double plasmid system that encodes T7 RNA polymerase (Tabor, S., and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078). Application of this expression system permitted exclusive radiolabeling of the cloned gene product, thereby providing a means to evaluate the level of expression and stability of encoded proteins. Fusion proteins were formed with the T7 bacteriophage gene 10 product and the 293 carboxyl-terminal residues of guanylate cyclase and two deletional mutants encoding 105 and 69 residues. Extracts prepared from bacteria expressing the carboxyl region, but not those expressing further deletions in this region, had substantial guanylate cyclase activity. There was no associated adenylate cyclase activity, suggesting that the catalytic domain retained its enzymatic specificity. These results provide direct evidence that the carboxyl portion of the membrane form of guanylate cyclase contains a catalytic domain. Homologous regions of the soluble form of guanylate cyclase and adenylate cyclase are likely to have enzymatic properties.
Thorpe
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DS
University Heart Center, University of Arizona College of Medicine, Tucson 85724.
Morkin
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D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006162:Guanylate Cyclase / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping
D011956:Receptors, Cell Surface; D009320:Atrial Natriuretic Factor; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor