Abstract
Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur.
Highlights
Transformation of chicken embryo fibroblasts by Rous receptors for stimulatory and inhibitory agonists.The catalyst of adenylate cyclase has only recently basal, Mg2+,and forskolin-stimulatedactivities; activities elicited by fluoride and guanosine 5’-0-(3-thiotriphosphate) are unaltered
The level of the catalytic componentof adenylate cyclase, assessed withactivated stimulatory guanine nucleotide-binding protein been purified to near homogeneity and appears to be a monomer having a molecular weight of 150,000 [12]
Inhibition of adenylate cyclase by guanine nucleotides and certain inhibitory agonists is likely to be effected by dissociation of Gi and consequent interaction of the Py heterodimer with the a subunit of G, interaction of the a subunit of Gi labeled NAD into 39,000- and 41,000-dalton proteins; with the catalyst, or interactions of both types [21,22,23,24]
Summary
Activities of adenylate cyclase were determined for membranes of untransformed and transformed CEF in the absence or presence of the indicated agents. Values represent the quotients (fS.E.) of the specific activities of adenylate cyclase for membranes of transformed CEF divided by those for membranes of untransformed C E F n denotes the number of embryos from which cells, membranes were derived. Growth medium was or was not replaced for untransformed CEF 16 h prior to harvesting of cells for preparation of membrane fractions; medium was always replaced for transformed cells as discussed under "Experimental Procedures."
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