Purification and characterization of a soluble form of mammalian adenylyl cyclase.
An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 micromol/min/mg, corresponding to a turnover number of 720 min-1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by Gsalpha and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein betagamma subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.
Dessauer
C W
CW
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
Gilman
A G
AG
D000256:Adenoviridae / Q000235:genetics; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D005576:Colforsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity
D000067956:Adenylyl Cyclase Inhibitors; D009838:Oligodeoxyribonucleotides; D011480:Protease Inhibitors; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000262:Adenylyl Cyclases