Identification and Properties of a Gs Protein in Catfish Liver Membranes

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 pmol/mg protein for [35S]GTPγS binding to catfish liver membranes were determined. The binding of the tracer was enhanced by 1 μM epinephrine, up to a maximum of 158%, and inhibited by NF 449, a Gsα-selective antagonist with half-maximal effect in the micromolar range. Immunoblotting analysis with a specific anti-Gsα antibody revealed a single band of about 45 kDa mass. This result represents the first demonstration of the presence of G protein αs subunits in the liver of an ectothermal vertebrate.