Molecular resolution and reconstitution of the GPP(NH) and NAF sensitive adenylate cyclase system

Abstract

When a special detergent-extraction procedure is applied to rat brain particulate fractions, the latter's adenylate cyclase activity becomes virtually unresponsive to NaF or Gpp (NH)p (guanylyl-5′-imidodiphosphate) despite the fact that under these conditions the enzyme does not appear to be removed (i.e., solubilized) from the membranes. Addition of exogenous fractions of detergent-solubilized membranes or of water-soluble samples of homogenates, obtained from various tissues, restores the stimulation of the enzyme by both Gpp(NH)p and NaF. These findings indicate that the stimulation caused by these agents is mediated by one or more regulatory component(s), and that these are molecular components physically distinct from the enzyme itself. The regulatory component(s) appear to be proteinaceous in nature and sensitive to SH-reactive reagents. The properties of the reconstituted system resemble those of the original particulate adenylate cyclase. This system may serve as a convenient tool for the study of the molecular properties of adenylate cyclase and of the basis of its regulatory control.