Solubilization of adenylyl cyclase from human myometrium in a alphas-coupled form.
Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5'-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrol-solubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Galphas large (52.2 kDa) and Galphas small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both alphas subunits of G proteins in human myometrium.
Bajo
Ana M
AM
Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain.
Prieto
Juan C
JC
Valenzuela
Pedro
P
Martinez
Pilar
P
Guijarro
Luis G
LG
D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism*; D002793:Cholic Acids / Q000737:chemistry; D002847:Chromatography, Agarose / Q000379:methods; D003902:Detergents / Q000737:chemistry; D005260:Female; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D009215:Myometrium / Q000201:enzymology; D017830:Octoxynol / Q000737:chemistry; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000737:chemistry; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D000077423:Polidocanol; D011092:Polyethylene Glycols; D017830:Octoxynol; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate