Beta subunit copurifies with GppNHp-activated adenylyl cyclase.

Area of Science:

• Biochemistry
• Molecular Biology
• Signal Transduction

Background:

• Previous studies suggested Gs (or alpha s) remains bound to adenylyl cyclase's catalytic subunit (C) during activation.
• The role of beta gamma subunits in the adenylyl cyclase activation cycle was not fully understood.

Purpose of the Study:

• To purify and characterize the GppNHp-activated bovine brain adenylyl cyclase complex.
• To investigate the association of beta gamma subunits with the activated adenylyl cyclase complex.

Main Methods:

• Purification of adenylyl cyclase using mild solution conditions and forskolin-agarose affinity chromatography.
• Analysis of subunit stoichiometry and complex dissociation using molecular-sieving chromatography (Superose 12 FPLC) and ultracentrifugation.

Main Results:

• GppNHp-activated adenylyl cyclase was purified over 3000-fold.
• The enzyme retained beta gamma subunits when bound to a forskolin-agarose column, even when activated, unless exposed to high salt.
• The stoichiometry of alpha s to beta to C was approximately 1:1:1, indicating beta gamma subunits do not dissociate from Gs upon activation.

Conclusions:

• Beta gamma subunits remain associated with the alpha s subunit of adenylyl cyclase throughout the activation cycle under specific conditions.
• Partial dissociation of beta gamma subunits from the complex occurs under conditions of high dilution or prolonged ultracentrifugation.
• An alternative model is proposed to explain the inhibitory effects of added beta gamma subunits on cyclase activity in platelet membranes.