Identification and properties of a Gs protein in catfish liver membranes.
The presence of G proteins and their involvement in adrenergic signaling has been investigated in catfish (Ictalurus melas) liver membranes. Adenylyl cyclase activity was potently stimulated by the nonhydrolyzable analog of GTP, [35S]guanosine 5'-O-(gamma-thiotriphosphate) (GTPgammaS) (maximal activation of about eightfold at 10(-5) M; half-maximal activation at 1.31 x 10(-7) M), and reduced by the competitive inhibitor of GTP, GDPbetaS (70% maximal inhibition at 10(-4) M; half-maximal inhibition at 1.98 x 10(-7) M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 microM), and its action was not affected by guanine nucleotides, confirming that the diterpene effect occurred only at targets downstream of the G proteins. Receptor-dependent G protein activity was evaluated by a [(35)S]GTPgammaS binding assay. At 100 microM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20 degrees, a Kd of 18.6 nM and a Bmax of 105.7 pmol/mg protein for [35S]GTPgammaS binding to catfish liver membranes were determined. The binding of the tracer was enhanced by 1 microM epinephrine, up to a maximum of 158%, and inhibited by NF 449, a G(s)alpha-selective antagonist with half-maximal effect in the micromolar range. Immunoblotting analysis with a specific anti-G(s)alpha antibody revealed a single band of about 45 kDa mass. This result represents the first demonstration of the presence of G protein alpha(s) subunits in the liver of an ectothermal vertebrate.
Fabbri
Elena
E
Department of Biology, University of Bologna, 40100 Bologna, Italy. ele@dns.unife.it
Selva
Corrado
C
Piano
Annamaria
A
Caselli
Federico
F
Capuzzo
Antonio
A
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D005576:Colforsin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism; D007059:Ictaluridae / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008566:Membranes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
D008024:Ligands; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D004837:Epinephrine