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-(γ-thiotriphosphate) (GTPγS) (maximal activation of about eightfold at 10-5 M; half-maximal activation at 1.31 × 10-7 M), and reduced by the competitive inhibitor of GTP, GDPβS (70% maximal inhibition at 10-4 M; half-maximal inhibition at 1.98 ×10-7 M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 μM), 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 [35S]GTPγS binding assay. At 100 μM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20°, a Kd of 18.6 nM and a Bmax of 105.7 pmo1/mg protein for [35S]GTPγS binding to catfish liver membranes wer...
Identification and properties of a Gs protein in catfish liver membranes
CAPUZZO, Antonio
2002
Abstract
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-(γ-thiotriphosphate) (GTPγS) (maximal activation of about eightfold at 10-5 M; half-maximal activation at 1.31 × 10-7 M), and reduced by the competitive inhibitor of GTP, GDPβS (70% maximal inhibition at 10-4 M; half-maximal inhibition at 1.98 ×10-7 M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 μM), 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 [35S]GTPγS binding assay. At 100 μM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20°, a Kd of 18.6 nM and a Bmax of 105.7 pmo1/mg protein for [35S]GTPγS binding to catfish liver membranes wer...I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
