The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution, activity, and properties of the 35,000-dalton (beta) subunit - PubMed

• PMID: 6309843

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The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution, activity, and properties of the 35,000-dalton (beta) subunit

J K Northup et al. J Biol Chem. 1983.

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Abstract

The stimulatory guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase is activated by exposure to guanine nucleotide analogs or to Al3+ + F-. Activated G/F can reconstitute adenylate cyclase activity when mixed with the catalytic moiety of the enzyme system in the absence of an effective free concentration of stimulatory ligand. Activation is explained by dissociation of the alpha (45,000-Da) and beta (35,000-Da) subunits of G/F. The beta subunit of G/F facilitates reversal of the activated state of the regulatory protein. This phenomenon, which has been exploited as an assay for the resolved beta subunit, has the following properties. 1) Addition of the resolved beta subunit to fluoride-activated G/F increases the initial rate of deactivation from a t 1/2 of 10 min to less than 0.5 min. 2) The enhancement of the rate of deactivation is a saturable process with a K 1/2 value of 60 ng/ml (approximately 2 nM). 3) G/F does not display beta subunit activity unless the alpha subunit has been inactivated or the subunits have been resolved. beta Subunit activity is measurable in detergent extracts of rabbit liver membranes or plasma membranes from S49 cell clones. The activity in such extracts is similar to that found with purified G/F, in that incubation at 30 degrees C in the presence of Mg2+ is required for its expression. However, cyc-, UNC, and H21a (S49 cell mutants with deficient or altered G/F activity) have amounts of beta subunit activity similar to that found in wild type S49 cells. Furthermore, the amount of beta subunit activity exceeds by 5- to 10-fold the amount expected based on the quantity of G/F in wild type extracts. All of the beta subunit activity in detergent extracts of liver membranes can be purified as a 35,000-Da polypeptide that is indistinguishable from the beta subunit of G/F. The beta subunit activity in extracts of cyc- membranes is expressed after incubation with guanine nucleotide analogs, implying association of the beta subunit with a GTP-binding protein. By analysis of the chromatographic behavior of G/F and the recently identified 41,000/35,000-Da heterodimeric substrate for the islet-activating protein from Bordetella pertussis, we have identified the 41,000-Da subunit of the substrate for islet-activating protein as the GTP-binding protein with which the majority of the beta subunit activity associates. These data have direct bearing on the mechanisms of hormonal activation and inhibition of adenylate cyclase.

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