Expression, purification, and assay of cytosolic (catalytic) domains of membrane-bound mammalian adenylyl cyclases.
The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes. They have permitted detailed mechanistic investigation of the actions of forskolin, Gs alpha, and the inhibitory G protein, Gi alpha. They have served as critical reagents for the development of plausible models of the catalytic mechanism of the enzyme. They have enabled X-ray crystallographic analysis of adenylyl cyclase; this technique was not approachable with the small quantities of the membrane-bound enzyme available previously. The information obtained by using the soluble domains of adenylyl cyclase has provided templates for description of the behavior of many forms of purine nucleotide cyclases from a variety of species. We now appreciate both adenylyl cyclases and guanylyl cyclases as dimeric enzymes with a 2-fold symmetrical domain arrangement (or pseudosymmetrical in the case of heterodimerization). The active sites are located at the interface between the two domains, both of which contribute binding surfaces.
Hatley
Mark E
ME
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Gilman
Alfred G
AG
Sunahara
Roger K
RK
D000262:Adenylyl Cyclases / Q000032:analysis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D000818:Animals; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D005576:Colforsin / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008566:Membranes / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D011994:Recombinant Proteins; D005576:Colforsin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases