Reconstitution of adenylate cyclase in Neurospora from two components of the enzyme

Abstract

The ATP-Mg2+-dependent, guanine nucleotide-stimulated adenylate cyclase of Neurospora crassa was shown to be composed of distinct components which, when mixed, can reconstitute a holoenzyme. After brief heat treatment, the adenylate cyclase activity in crude homogenates of Neurospora was found to have reduced activity and greatly reduced sensitivity to stimulation by guanyl-5'-yl-imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity could be restored by addition of a homogenate from a crisp-1 (cr-1) mutant of Neurospora which was shown to have little or no adenylate cyclase activity. These results show that the adenylate cyclase of Neurospora resembles the enzyme of animal systems in that it is composed of distinct regulatory and catalytic components. The data presented also show that the cr-1 mutant is defective in the adenylate cyclase catalytic component but not in the regulatory component. Therefore, cr-1 is unlike most other such eucaryotic mutants which appear to be defective in the regulatory component.